Essential fungal genes and their use

ABSTRACT

Abstract of the Disclosure Disclosed are essential Aspergillus polypeptides and genes (AN97, AN17, AN80, and AN85), as well as homologs thereof, which can be used to identify antifungal agents for treating fungal infections such as aspergillosis.

BACKGROUND OF THE INVENTION

[0001] The invention relates to essential fungal genes and their use inidentifying antifungal agents.

[0002] Fungal infections (mycoses) may be cutaneous, subcutaneous, orsystemic. Superficial mycoses include tinea capitis, tinea corporis,tinea pedis, perionychomycosis, pityriasis versicolor, oral thrush, andother candidoses such as vaginal, respiratory tract, biliary,eosophageal, and urinary tract candidoses. Systemic mycoses includesystemic and mucocutaneous candidosis, cryptococcosis, aspergillosis,mucormycosis (phycomycosis), paracoccidioidomycosis, North Americanblastomycosis, histoplasmosis, coccidioidomycosis, and sporotrichosis.Fungal infections can also contribute to meningitis and pulmonary orrespiratory tract diseases. Opportunistic fungal infections proliferate,especially in patients afflicted with AIDS or other diseases thatcompromise the immune system.

[0003] Examples of pathogenic fungi include dermatophytes (e.g.,Microsporum canis and other M. spp.; and Trichophyton spp. such as T.rubrum, and T. mentagrophytes), yeasts (e.g., Candida albicans, C.Tropicalis, or other Candida species), Torulopsis glabrata,Epidermophyton floccosum, Malassezia furfur (Pityropsporon orbiculare,or P. ovale), Cryptococcus neoformans, Aspergillus fumigatus, and otherAspergillus sp., Zygomycetes (e.g., Rhizopus, Mucor), Paracoccidioidesbrasiliensis, Blastomyces dermatitides, Histoplasma capsulatum,Coccidioides immitis, and Sporothrix schenckii.

[0004] Various strains of the fungus Aspergillus sp. causeaspergillosis, a potentially life-threatening disease in humans andother mammals. The clinical manifestations of aspergillosis in humansare very similar to those observed in rodents and cows. For example,necrosis, angioinvasion, and hematogenous dissemination are commonfeatures of aspergillosis in rodent and bovine model systems and inhumans. In humans, aspergillosis typically is caused by inhalation ofconidia (i.e., asexual spores produced by the fungus). In cattle,pathogenic Aspergillus typically enter the animal through theforestomach and then disseminate through the blood of the animal.Putative virulence factors produced by pathogenic species of Aspergillusinclude hydroxymate siderophores (i.e., compounds that compete withhuman iron-binding proteins to acquire iron to support fungal growth),lipids having the ability to inhibit complement and phagocytosis, andproteinases that can degrade elastin and other substrates.

SUMMARY OF THE INVENTION

[0005] The invention is based on the discovery of four new genes in thefungus Aspergillus nidulans that are essential for survival. These genesare referred to herein as AN97, AN80, AN17, and AN85; for convenience,the polypeptides encoded by these genes are referred to herein as “ANpolypeptides.” The genes encoding the AN polypeptides are usefulmolecular tools for identifying similar genes in pathogenicmicrorganisms, such as pathogenic strains of Aspergillus (e.g.Aspergillus fumigatus and Aspergillus flavus). In addition, the ANpolypeptides and the essential genes encoding them are useful targetsfor identifying compounds that are inhibitors of the pathogens in whichthe AN polypeptides are expressed. Such inhibitors inhibit fungal growthby being fungistatic (e.g., inhibiting reproduction or cell division) orby being fungicidal (i.e., by causing cell death).

[0006] The invention, therefore, features an isolated AN97 polypeptidehaving the amino acid sequence set forth in SEQ ID NO:1, or conservativevariations thereof. Nucleic acids encoding AN97 also are included withinthe invention. In particular, the invention includes an isolated nucleicacid of (a) SEQ ID NO:2, as depicted in FIG. 1, or degenerate variantsthereof; (b) SEQ ID NO:2, or degenerate variants thereof, wherein T isreplaced by U; (c) nucleic acids complementary to (a) and (b); and (d)fragments of (a), (b), and (c) that are at least 15 base pairs in lengthand that hybridize under stringent conditions to genomic DNA encodingthe polypeptide of SEQ ID NO:1.

[0007] The invention also features an isolated AN80 polypeptide havingthe amino acid sequence set forth in SEQ ID NO:3, or conservativevariations thereof. Nucleic acids encoding AN80 also are included. Inparticular, the invention includes an isolated nucleic acid of: (a) SEQID NO:4, as depicted in FIG. 2, or degenerate variants thereof; (b) SEQID NO:4, or degenerate variants thereof, wherein T is replaced by U; (c)nucleic acids complementary to (a) and (b); and (d) fragments of (a),(b), and (c) that are at least 15 base pairs in length and whichhybridize under stringent conditions to genomic DNA encoding thepolypeptide of SEQ ID NO:3.

[0008] The invention also includes an isolated AN85 polypeptide havingthe amino acid sequence set forth in SEQ ID NO:5, or conservativevariations thereof. Nucleic acids encoding AN85 also are included. Inparticular, the invention includes an isolated nucleic acid of: (a) SEQID NO:6, as depicted in FIG. 3, or degenerate variants thereof; (b) SEQID NO:6, or degenerate variants thereof, wherein T is replaced by U; (c)nucleic acids complementary to (a) and (b); and (d) fragments of (a),(b), and (c) that are at least 15 base pairs in length and whichhybridize under stringent conditions to genomic DNA encoding thepolypeptide of SEQ ID NO:5.

[0009] The invention also features an isolated AN17 polypeptide havingthe amino acid sequence set forth in SEQ ID NO:7, or conservativevariations thereof. Nucleic acids encoding AN17 also are included. Inparticular, the invention includes an isolated nucleic acid of: (a) SEQID NO:8, as depicted in FIG. 4, or degenerate variants thereof; (b) SEQID NO:8, or degenerate variants thereof, wherein T is replaced by U; (c)nucleic acids complementary to (a) and (b); and (d) fragments of (a),(b), and (c) that are at least 15 base pairs in length and whichhybridize under stringent conditions to genomic DNA encoding thepolypeptide of SEQ ID NO:7.

[0010] The invention also includes isolated nucleic acids that are atleast 15 base pairs in length and which hybridize under stringentconditions to a nucleotide sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. In addition,the invention includes allelic variants (i.e., genes encoding isozymes)of the genes encoding AN97, AN17, AN80, and AN85. For example, theinvention includes genes that encode an AN polypeptide but which geneincludes point mutation, deletion, promoter variant, or splice sitevariant, provided that the resulting AN polypeptide functions as an ANpolypeptide (e.g., as determined in a complementation assay, asdescribed herein and elsewhere). Also included within the invention areisolated nucleic acid molecules containing the cDNA sequences containedwith ATCC accession numbers ______, ______, ______, and ______, as wellas polypeptides encoded by the cDNA sequences of these nucleic acidmolecules.

[0011] Identification of the AN97, AN17, AN80, and AN85 genes and thedetermination that they are essential allows homologs of these genes tobe found in other organisms (e.g., fungi, such as yeast like S.cerevisiae; mammalian cells, such as human or murine cells; or plantcells). Thus, the AN polypeptides used not only can be as a model foridentifying similar essential genes in other Aspergillus strains, butalso to identify homologous essential genes in other organisms, e.g., S.cerevisiae. Because such genes are homologs, they can be expected to beessential for survival without the need for extensive characterizationof the homologous gene or polypeptide. Even though some such homologousgenes may have previously been identified, the invention allows one todetermine that such genes are essential for survival. Having identifiedsuch homologous genes as essential, these genes and the polypeptidesencoded by these genes can be used to identify compounds that inhibitthe growth of the host organism (e.g., compounds that are fungicidal orfungistatic against pathogenic strains of the organism).

[0012] As used herein, the term “yeast” refers to organisms of the orderSaccharomycetales, which includes yeast such as Saccharomyces andCandida. As described below, several homologs of the AN polypeptideshave been identified in the yeast S. cerevisiae and are essential forsurvival. Given the identification of such genes as essential in S.cerevisiae, homologs of these essential yeast genes can also be found inpathogenic yeast strains (e.g., Candida albicans). The S. cerevisiaepolypeptide and gene termed D9798.4 are homologs of the AN97 polypeptideand gene. The D9798.4 polypeptide and nucleic acid are depicted in FIG.5, and are set forth in SEQ ID NOs:9 and 10, respectively (GenBankAccession No. U32517). As described herein, various methods of theinvention can utilize the D9798.4 polypeptide or conservative variationsthereof. Also useful are isolated nucleic acids of (a) SEQ ID NO:10, asdepicted in FIG. 5, or degenerate variants thereof; (b) SEQ ID NO:10, ordegenerate variants thereof, wherein T is replaced by U; (c) nucleicacids complementary to (a) and (b); and (d) fragments of (a), (b), and(c) that are at least 15 base pairs in length and which hybridize understringent conditions to genomic DNA encoding the polypeptide of SEQ IDNO:9.

[0013] Yeast homologs of the AN85 and AN80 polypeptides and genes alsohave been identified as being essential for survival, and these homologscan be used in the methods described herein. As described above forAN97, conservative variations, degenerate variants, complementarysequences, fragments, and nucleic acids in which T is replaced by U alsocan be used in various methods of the invention. Two homologs of AN85have been identified. The amino acid and nucleic acid sequences of theAN85 homolog termed YGR010W are depicted in FIG. 6 (GenBank AccessionNo. Z72795); these sequences are set forth as SEQ ID NOs:11 and 12,respectively. The amino acid and nucleic acid sequences of the AN85homolog termed L8543.16 are depicted in FIG. 7 (GenBank Accession No.U20618); these sequences are set forth as SEQ ID NOs:13 and 14,respectively. The AN80 polypeptide and gene have a homolog in yeast,termed L8004.2, the amino acid and nucleic acid sequences of which aredepicted in FIG. 8 (GenBank Accession No. U53876). These sequences areset forth as SEQ ID NOs:15 and 16, respectively.

[0014] The term AN97 polypeptide or gene as used herein is intended toinclude the polypeptide and gene set forth in FIG. 1 herein, as well ashomologs of the sequences set forth in FIG. 1. For example, encompassedby the term AN97 gene are degenerate variants of the nucleic acidsequence set forth in FIG. 1. (SEQ ID NO:2). Degenerate variants of anucleic acid sequence exist because of the degeneracy of the amino acidcode; thus, those sequences that vary from the sequence represented bySEQ ID NO:2, but which nonetheless encode an AN97 polypeptide areincluded within the invention. Likewise, because of the similarity inthe structures of amino acids, conservative variations can be made inthe amino acid sequence of the AN97 polypeptide while retaining thefunction of the polypeptide (e.g., as determined in a complementationassay, as described herein and elsewhere). AN97 polypeptides and genesidentified in additional Aspergillus strains may be such conservativevariations or degenerate variants of the particular AN97 polypeptide andnucleic acid set forth in FIG. 1 (SEQ ID NOs:1 and 2, respectively). TheAN97 polypeptide and gene share at least 80%, e.g., 90%, sequenceidentity with SEQ ID NOs:1 and 2, respectively. Regardless of thepercent sequence identity between the AN97 sequence and the sequencerepresented by SEQ ID NOs:1 and 2, the AN97 genes and polypeptidesencompassed by the invention are able to complement for the lack of AN97function (e.g., in a temperature-sensitive mutant) in a standardcomplementation assay. AN97 genes that are identified and cloned fromadditional Aspergillus strains, and pathogenic strains in particular,can be used to produce AN97 polypeptides for use in the various methodsdescribed herein, e.g., for identifying antifungal agents. Likewise, theterm AN80 encompasses homologues and conservative and degeneratevariants of the sequences depicted in FIG. 2. Such homologues,conservative variations, and degenerate variants of AN17, AN85, and AN80also are included within the invention. Excluded from the invention arethe naturally-occurring homologs of AN polypeptides and nucleic acidsfound in S. cerevisiae (D9798.4, L8543.16, YGR010W, and L8004.2),although methods employing such polypeptides and nucleic acids areencompassed by the invention.

[0015] The AN97, AN17, AN80, and AN85 genes have been identified andshown to be essential for survival, these AN polypeptides and theiryeast homologs (e.g., D9798.4, L8543.16, YGR010W, and L8004.2) can beused to identify antifungal agents. More specifically, these ANpolypeptides and their yeast homologs can be used, separately ortogether, in assays to identify test compounds which bind thesepolypeptides. Such test compounds are expected to be antifungal agents,in contrast to compounds that do not bind AN97, AN17, AN80, AN85,D9798.4, L8543.16, YGR010W, and/or L8004.2. As described herein, any ofa variety of art-known methods can be used to assay for binding of testcompounds to the polypeptides. The invention includes, for example, amethod for identifying an antifungal or anti-yeast agent where themethod entails: (a) contacting an AN polypeptide, or homolog thereof,with a test compound; (b) detecting binding of the test compound to theAN polypeptide or homolog; and (c) determining whether a test compoundthat binds the AN polypeptide or homolog inhibits growth of fungi oryeast, relative to growth of fungi or yeast cultured in the absence ofthe test compound that binds the AN polypeptide or homolog, as anindication that the test compound is an antifungal or anti-yeast agent.

[0016] In various embodiments, the AN polypeptide is derived from anon-pathogenic or pathogenic Aspergillus strain, such as Aspergillusnidulans, Aspergillus fumigatus, Aspergillus flavus, and Aspergillusniger. Preferably, homologs thereof are derived from the yeastSaccharomyces cerevisiae. The test compound can be immobilized on asubstrate, and binding of the test compound to the AN polypeptide orhomolog can be detected as immobilization of the AN polypeptide orhomolog on the immobilized test compound, e.g., in an immunoassay withan antibody that specifically binds AN97.

[0017] If desired, the test compound can be a test polypeptide (e.g., apolypeptide having a random or predetermined amino acid sequence; or anaturally-occurring or synthetic polypeptide). Alternatively, the testcompound can be a nucleic acid, such as a DNA or RNA molecule. Inaddition, small organic molecules can be tested. The test compound canbe a naturally-occurring compound or it can be synthetically produced,if desired. Synthetic libraries, chemical libraries, and the like can bescreened to identify compounds that bind the AN polypeptides. Moregenerally, binding of test compound to the AN polypeptide or homolog canbe detected either in vitro or in vivo. Regardless of the source of thetest compound, the AN polypeptides described herein can be used toidentify compounds that are fungicidal or fungistatic to a variety ofpathogenic or non-pathogenic strains.

[0018] In an exemplary method, binding of a test compound to an ANpolypeptide can be detected in a conventional two-hybrid system fordetecting protein/protein interactions (e.g., in yeast or mammaliancells). Generally, in such a method, (a) the AN polypeptide is providedas a fusion protein that includes the AN polypeptide fused to (i) atranscription activation domain of a transcription factor or (ii) aDNA-binding domain of a transcription factor; (b) the test polypeptideis provided as a fusion protein that includes the test polypeptide fusedto (i) a transcription activation domain of a transcription factor or(ii) a DNA-binding domain of a transcription factor; and (c) binding ofthe test polypeptide to the AN polypeptide polypeptide is detected asreconstitution of a transcription factor. The yeast homologs can be usedin similar methods. Reconstitution of the transcription factor can bedetected, for example, by detecting transcription of a gene that isoperably linked to a DNA sequence bound by the DNA-binding domain of thereconstituted transcription factor (See, for example, White, 1996, Proc.Natl. Acad. Sci. 93:10001-10003 and references cited therein and Vidalet al., 1996, Proc. Natl. Acad. Sci. 93:10315-10320).

[0019] In an alternative method, an isolated nucleic acid moleculeencoding an AN polypeptides is used to identify a compound thatdecreases the expression of the AN polypeptide in vivo. Such compoundscan be used as antifungal agents. To discover such compounds, cells thatexpress an AN polypeptide are cultured, exposed to a test compound (or amixture of test compounds), and the level of expression or activity iscompared with the level of AN polypeptide expression or activity incells that are otherwise identical but that have not been exposed to thetest compound(s). Many standard quantitative assays of gene expressioncan be utilized in this aspect of the invention.

[0020] In order to identify compounds that modulate expression of an ANpolypeptide (or homologous sequence), the test compound(s) can be addedat varying concentrations to the culture medium of cells that express anAN polypeptide (or homolog), as described above. Such test compounds caninclude small molecules (typically, non-protein, non-polysaccharidechemical entities), polypeptides, and nucleic acids. The expression ofthe AN polypeptide is then measured, for example, by Northern blot PCRanalysis or RNAse protection analyses using a nucleic acid molecule ofthe invention as a probe. The level of expression in the presence of thetest molecule, compared with the level of expression in its absence,will indicate whether or not the test molecule alters the expression ofthe AN polypeptide. Because the AN polypeptides are essential forsurvival, test compounds that inhibit the expression and/or function ofthe AN polypeptide will inhibit growth of the cells or kill the cells.

[0021] Compounds that modulate the expression of the polypeptides of theinvention can be identified by carrying out the assay described aboveand then measuring the levels of the AN polypeptides expressed in thecells, e.g., by performing a Western blot analysis using antibodies thatbind an AN polypeptide.

[0022] The invention further features methods of identifying from alarge group of mutants those strains that have conditional lethalmutations. In general, the gene and corresponding gene product aresubsequently identified, although the strains themselves can be used inscreening or diagnostic assays. The mechanism(s) of action for theidentified genes and gene products provide a rational basis for thedesign of anti-fungal therapeutic agents. These antifungal agents reducethe action of the gene product in a wild type strain, and therefore areuseful in treating a subject with that type, or a similarly susceptibletype of infection by administering the agent to the subject in apharmaceutically effective amount. Reduction in the action of the geneproduct includes competitive inhibition of the gene product for theactive site of an enzyme or receptor; non-competitive inhibition;disrupting an intracellular cascade path which requires the geneproduct; binding to the gene product itself, before or afterpost-translational processing; and acting as a gene product mimetic,thereby down-regulating the activity. Therapeutic agents includemonoclonal antibodies raised against the gene product.

[0023] Furthermore, the presence of the gene sequence in certain cells(e.g., a pathogenic fungus of the same genus or similar species), andthe absence or divergence of the sequence in host cells can bedetermined, if desired. Therapeutic agents directed toward genes or geneproducts that are not present in the host have several advantages,including fewer side effects, and lower overall dosage.

[0024] The invention includes pharmaceutical formulations that include apharmaceutically acceptable excipient and an antifungal agent identifiedusing the methods described herein. In particular, the inventionincludes pharmaceutical formulations that contain antifungal agents thatinhibit the growth of, or kill, pathogenic Aspergillus strains. Suchpharmaceutical formulations can be used for treating an Aspergillusinfection in an organism. Such a method entails administering to theorganism a therapeutically effective amount of the pharmaceuticalformulation. In particular, such pharmaceutical formulations can be usedto treat aspergillosis in mammals such as humans and domesticatedmammals (e.g., cows and pigs). The efficacy of such antifungal agents inhumans can be estimated in an animal model system well known to those ofskill in the art (e.g., bovine and rodent (e.g., mouse) model systems).These formulations also can be used to treat fungal infections inplants, e.g., by topically applying the antifungal agent to the plant.Alternatively, where the antifungal agent is a polypeptide or anantisense RNA, a gene encoding the polypeptide or expressing theantisense RNA can be transfected into the plant, using conventionaltechniques, and the polypeptide or antisense RNA can be expressed in theplant.

[0025] Also included within the invention are polyclonal and monoclonalantibodies that specifically bind AN97, AN17, AN80, or AN85 polypeptide.Such antibodies can facilitate detection of AN polypeptides in variousAspergillus strains. These antibodies also are useful for detectingbinding of a test compound to AN97, AN17, AN80, or AN85 polypeptides(e.g., using the assays described herein). In addition, monoclonalantibodies that bind AN97, AN17, AN80, or AN85 polypeptide arethemselves adequate antifungal agents when administered to a mammal, assuch monoclonal antibodies are expected to impede one or more functionsof AN97, AN17, AN80, or AN85 polypeptide.

[0026] As used herein, “nucleic acids” encompass both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid may be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid may be a sensestrand or an antisense strand. The nucleic acid may be synthesized usingoligonucleotide analogs or derivatives (e.g., inosine orphosphorothioate nucleotides). Such oligonucleotides can be used, forexample, to prepare nucleic acids that have altered base-pairingabilities or increased resistance to nucleases.

[0027] An “isolated nucleic acid” is a DNA or RNA that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.Thus, in one embodiment, an isolated nucleic acid includes some or allof the 5′ non-coding (e.g., promoter) sequences that are immediatelycontiguous to the coding sequence. The term therefore includes, forexample, a recombinant DNA that is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other sequences. It also includesa recombinant DNA that is part of a hybrid gene encoding an additionalpolypeptide sequence. The term “isolated” can refer to a nucleic acid orpolypeptide that is substantially free of cellular material, viralmaterial, or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an “isolated nucleic acid fragment” is a nucleicacid fragment that is not naturally occurring as a fragment and wouldnot be found in the natural state.

[0028] A nucleic acid sequence that is “substantially identical” to anAN97, AN17, AN80, or AN85 nucleotide sequence is at least 80% or 85%identical to the nucleotide sequence of the Aspergillus AN97, AN80,AN85, and AN17 nucleic acids of SEQ ID NO:2, NO:4, NO:6, and NO:8,respectively, as depicted in FIGS. 1, 2, 3, and 4, respectively. Forpurposes of comparison of nucleic acids, the length of the referencenucleic acid sequence will generally be at least 40 nucleotides, e.g.,at least 60 nucleotides or more nucleotides. Sequence identity can bemeasured using sequence analysis software (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

[0029] The AN polypeptides of the invention include, but are not limitedto, recombinant polypeptides and natural polypeptides. The inventionalso encompasses nucleic acid sequences that encode forms of AN97, AN17,AN80, or AN85 polypeptides in which naturally occurring amino acidsequences are altered or deleted. Preferred nucleic acids encodepolypeptides that are soluble under normal physiological conditions.Also within the invention are nucleic acids encoding fusion proteins inwhich a portion of AN97, AN17, AN80, or AN85 is fused to an unrelatedpolypeptide (e.g., a marker polypeptide or a fusion partner) to create afusion protein. For example, the polypeptide can be fused to ahexa-histidine tag to facilitate purification of bacterially expressedpolypeptides, or to a hemagglutinin tag to facilitate purification ofpolypeptides expressed in eukaryotic cells. The invention also includesisolated, for example, polypeptides (and the nucleic acids that encodethese polypeptides) that include a first portion and a second portion;the first portion includes, e.g., an AN polypeptide, and the secondportion includes an immunoglobulin constant (Fc) region or a detectablemarker.

[0030] The fusion partner can be, for example, a polypeptide whichfacilitates secretion, e.g., a secretory sequence. Such a fusedpolypeptide is typically referred to as a preprotein. The secretorysequence can be cleaved by the host cell to form the mature protein.Also within the invention are nucleic acids that encode AN97, AN17,AN80, or AN85 fused to a polypeptide sequence to produce an inactivepreprotein. Preproteins can be converted into the active form of theprotein by removal of the inactivating sequence.

[0031] The invention also includes nucleic acids that hybridize, e.g.,under stringent hybridization conditions (as defined herein) to all or aportion of the nucleotide sequence of SEQ ID NOs: 2, 4, 6, or 8, ortheir complements. The hybridizing portion of the hybridizing nucleicacids is typically at least 15 (e.g., 20, 30, or 50) nucleotides inlength. The hybridizing portion of the hybridizing nucleic acid is atleast 80%, e.g., at least 95%, or at least 98%, identical to thesequence of a portion or all of a nucleic acid encoding an AN97, AN17,AN80, or AN85 polypeptide. Hybridizing nucleic acids of the typedescribed herein can be used as a cloning probe, a primer (e.g., a PCRprimer), or a diagnostic probe. Nucleic acids that hybridize to thenucleotide sequences of SEQ ID NOs:2, 4, 6, or 8 are considered“antisense oligonucleotides.” Also included within the invention areribozymes that inhibit the function of AN97, AN17, AN80, or AN85, asdetermined, for example, in a complementation assay.

[0032] In another embodiment, the invention features cells, e.g.,transformed host cells, that contain a nucleic acid encompassed by theinvention. A “transformed cell” is a cell into which (or into anancestor of which) has been introduced, by means of recombinant DNAtechniques, a nucleic acid encoding an AN polypeptide. Both prokaryoticand eukaryotic cells are included, e.g., bacteria, Aspergillus, yeast,and the like.

[0033] The invention also features genetic constructs (e.g., vectors andplasmids) that include a nucleic acid of the invention which is operablylinked to a transcription and/or translation sequence to enableexpression, e.g., expression vectors. By “operably linked” is meant thata selected nucleic acid, e.g., a DNA molecule encoding an ANpolypeptide, is positioned adjacent to one or more sequence elements,e.g., a promoter, which directs transcription and/or translation of thesequence such that the sequence elements can control transcriptionand/or translation of the selected nucleic acid.

[0034] The invention also features purified or isolated AN97, AN17,AN80, and AN85 polypeptides. As used herein, both “protein” and“polypeptide” mean any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). Thus, the terms “AN97 polypeptide” (or AN97), “AN17polypeptide” (or AN17), “AN80 polypeptide” (or AN80), or “AN85polypeptide” (or AN85) include full-length, naturally occurring AN97,AN17, AN80, or AN85 proteins, respectively, as well as recombinantly orsynthetically produced polypeptides that correspond to a full-length,naturally occurring AN97, AN17, AN80, or AN85 protein, or to a portionof a naturally occurring or synthetic AN97, AN17, AN80, or AN85polypeptide.

[0035] A “purified” or “isolated” compound is a composition that is atleast 60% by weight the compound of interest, e.g., an AN97 polypeptideor antibody. Preferably the preparation is at least 75% (e.g., at least90% or 99%) by weight the compound of interest. Purity can be measuredby any appropriate standard method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

[0036] Preferred AN97, AN17, AN80, AN85 polypeptides include a sequencesubstantially identical to all or a portion of a naturally occurringAN97, AN17, AN80, or AN85 polypeptide, e.g., including all or a portionof the sequences shown in FIGS. 1, 2, 3, and 4, respectively.Polypeptides “substantially identical” to the AN polypeptide sequencesdescribed herein have an amino acid sequence that is at least 80% or 85%(e.g., 90%, 95% or 99%) identical to the amino acid sequence of theAN97, AN80, AN85 or AN17 polypeptides of SEQ ID NOs:1, 3, 5, and 7,respectively. For purposes of comparison, the length of the reference ANpolypeptide sequence will generally be at least 16 amino acids, e.g., atleast 20 or 25 amino acids.

[0037] In the case of polypeptide sequences that are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, conservative substitutions for thereference sequence. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

[0038] Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference polypeptide. Thus, a polypeptidethat is 50% identical to a reference polypeptide that is 100 amino acidslong can be a 50 amino acid polypeptide that is completely identical toa 50 amino acid long portion of the reference polypeptide. It also mightbe a 100 amino acid long polypeptide which is 50% identical to thereference polypeptide over its entire length. Of course, otherpolypeptides also will meet the same criteria.

[0039] The invention also features purified or isolated antibodies thatspecifically bind to an AN polypeptide. By “specifically binds” is meantthat an antibody recognizes and binds a particular antigen, e.g., anAN97, AN17 polypeptide, but does not substantially recognize and bindother molecules in a sample, e.g., a biological sample that naturallyincludes AN97, AN17, AN80, or AN85. In one embodiment the antibody is amonoclonal antibody.

[0040] In another aspect, the invention features a method for detectingan AN polypeptide in a sample. This method includes: obtaining a samplesuspected of containing AN97, AN17, AN85, or AN80; contacting the samplewith an antibody that specifically binds an AN97, AN17, AN85 or AN80polypeptide under conditions that allow the formation of complexes of anantibody and AN97, AN17, AN85 or AN80; and detecting the complexes, ifany, as an indication of the presence of AN97, AN17, AN85 or AN80 in thesample.

[0041] Also encompassed by the invention is a method of obtaining a generelated to (i.e., a functional homologue of) the AN97, AN17, AN85, orAN80 gene. Such a method entails obtaining a labeled probe that includesan isolated nucleic acid which encodes all or a portion of AN97, AN17,AN85, or AN80, or a homolog thereof (e.g., D9798.4, L8543.16, YGR010W,or L8004.2); screening a nucleic acid fragment library with the labeledprobe under conditions that allow hybridization of the probe to nucleicacid fragments in the library, thereby forming nucleic acid duplexes;isolating labeled duplexes, if any; and preparing a full-length genesequence from the nucleic acid fragments in any labeled duplex to obtaina gene related to the AN97, AN17, AN85, or AN80 gene.

[0042] The invention offers several advantages. By combining geneknockout assays, as described herein, with assays of conditionalsensitivity, we have identified genes that are truly essential, i.e.,genes whose absence is fungicidal to Aspergillus. In addition, themethods for identifying antifungal agents can be configured for highthroughput screening of numerous candidate antifungal agents.

[0043] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated hereinby reference in their entirety. In the case of a conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative and are not intendedto limit the scope of the invention, which is defined by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 is a representation of the amino acid and nucleic acidsequences of the AN97 polypeptide and gene from an Aspergillus nidulansstrain (SEQ ID NOs:1 and 2, respectively).

[0045]FIG. 2 is a representation of the amino acid and nucleic acidsequences of the AN80 polypeptide and gene from an Aspergillus nidulansstrain (SEQ ID NOs:3 and 4, respectively).

[0046]FIG. 3 is a representation of the amino acid and nucleic acidsequences of the AN85 polypeptide and gene from an Aspergillus nidulansstrain (SEQ ID NOs:5 and 6, respectively).

[0047]FIG. 4 is a representation of the amino acid and nucleic acidsequences of the AN17 polypeptide and gene from an Aspergillus nidulansstrain (SEQ ID NOs:7 and 8, respectively).

[0048]FIG. 5 is a representation of the amino acid and nucleic acidsequences of the D9798.4 polypeptide and gene from S. cerevisiae (SEQ IDNOs:9 and 10, respectively).

[0049]FIG. 6 is a representation of the amino acid and nucleic acidsequences of the YGR010W polypeptide and gene from S. cerevisiae (SEQ IDNOs:11 and 12, respectively).

[0050]FIG. 7 is a representation of the amino acid and nucleic acidsequences of the L8543.16 polypeptide and gene from S. cerevisiae (SEQID NOs:12 and 13, respectively).

[0051]FIG. 8 is a representation of the amino acid and nucleic acidsequences of the L8004.2 polypeptide and gene from S. cerevisiae (SEQ IDNOs:14 and 15, respectively).

DETAILED DESCRIPTION OF THE INVENTION

[0052] Identifying Essential Aspergillus Genes

[0053] As shown by the experiments described below, expression of eachof the AN97, AN17, AN80, and AN85 polypeptides is essential for survivalof Aspergillus nidulans. Aspergillus nidulans is available from the ATCC(#FGSC4). To identify genes for which inhibition of gene expression isfungicidal, various mutants of Aspergillus nidulans were assayed forconditional sensitivity. In general, mutagenesis of Aspergillus nidulanscan be accomplished using any of various art-known methods. For example,exposure to ultraviolet light, x-rays, and/or chemical mutagens isacceptable. Examples of suitable chemical mutagens includeethylmethansulfonate (EMS), metyhlmethanesulfonate (MMS),methylnitrosoguanidine (NTG), 4-nitroquinoline-1-oxide (NQO),2-aminopurine, 5-bromouracil, ICR 191 and other acridine derivatives,sodium bisulfite, ethidium bromide, nitrous acid, hydroxylamine,N-methyl-N′-nitroso-N-nitroguanidine, and alkylating agents (for furtherdescription of art-known mutagens and mutagenesis methods, see, e.g.,Current Protocols in Molecular Biology, 1995 and Adelberg et al.,Biochem. Biophys. Res. Comm. 18:788, 1965).

[0054] To identify conditional-sensitive mutants, mutagenized cells canbe grown under (a) a first set of permissive conditions, then shifted to(b) restrictive conditions, and then to (c) a second set of permissiveconditions. The cells of interest are those mutants that grow under thepermissive conditions of (a), but fail to grow under the restrictiveconditions of (b), and fail to recover under the permissive conditionsof (c).

[0055] Ostensibly, any change in a growth parameter can serve as the“restrictive condition.” For example, the restrictive conditions may bemet by increasing or decreasing the temperature at which the cells aregrown, thereby allowing the identification of temperature-sensitivemutants. For example, the optimal growth temperature for A. nidulans is28° C., and a typical restrictive temperature is 42° C. In alternativemethods, the change to a restrictive condition may entail changing oneor more of the following parameters of the growth conditions: pH, typeand/or concentration of carbon and nitrogen sources, trace minerals,vitamins, salts, conidia-forming materials (e.g., DMSO, glycerol, anddeuterated water), humidity, and the like. In general, permissive growthconditions allow the strains to grow at a rate that is at least 75% ofthat of the wild-type growth rate of Aspergillus. The second set ofpermissive conditions (in (c)) can be the same as, or different from,the first permissive conditions. Typically, the cells are subjected tothe second permissive conditions for at least 2 growth cycles (moretypically, at least 5, 10, 15 or even 20 growth cycles). Generally, thecells are subjected to the restrictive conditions for 2 to 20 growthcycles (typically 2-10 growth cycles) and for 24 hours or less.

[0056] In practicing the invention, cell death (e.g., in (b)) can bedetected using any of a number of conventional criteria. For example,cell death can be detected macroscopically by observing that a colony ofcells has approximately the same size, or a reduced size, after a lengthof time that is normally sufficient for several growth cycles under thesecond permissive conditions. Detection of cell death also can befacilitated by the use of light microscopy and cell staining to revealcytological deformations and/or morphologies commonly known to beindicative of cell death. The absence of DNA, RNA, or protein synthesisalso can signify cell death.

[0057] Identification of Homologs of AN Polypeptides

[0058] Having shown that the AN97, and AN80, and AN85 genes andpolypeptides are essential for survival in Aspergillus, it can beexpected that homologs of these polypeptides, when present in otherorganisms, for example pathogenic yeast, are essential for survival ofthose organisms as well. Using the sequences of the AN polypeptidesidentified in Aspergillus, homologs of these polypeptides wereidentified in the yeast S. cerevisiae. The coding sequences of AN97,AN80, and AN85 were used to search the GenBank database of nucleotidesequences to identify homologs of AN97, AN80, and AN85, respectively,which are essential genes in other organisms. Sequence comparisons wereperformed using the Basic Local Alignment Search Tool (BLAST) (Altschulet al., J. Mol. Biol., 215:403-410 1990). The percent sequence identityshared by the AN polypeptides and their homologs were determined usingthe GAP program from the Genetics Computer Group (GCG) WisconsinSequence Analysis Package (Wisconsin Package Version 9.0, GCG; Madison,Wis.). The following parameters were used: gap creation penalty, 12(protein) 50 (DNA); gap extension penalty, 4 (protein) 3 (DNA). Thepercent sequence identity shared by the AN polypeptides and theirhomologs are summarized in Table 1. Typically, the AN polypeptides andtheir homologs share at least 25% (e.g., at least 40%) sequenceidentity. Typically, the DNA sequences encoding AN polypeptides andtheir homologs share at least 35% (e.g., at least 45%) sequenceidentity. TABLE 1 Sequence Identity Shared by AN Polypeptides and TheirHomologues. % Identity of DNA Sequences % Identity of Homolog in (codingPolypeptide AN Polypeptide Saccharomyces region) Sequences AN80 L8004.237.4 27.9 AN85 YGR010W 50.2 41.0 AN85 L8543.16 49.2 43.7 AN97 D9798.438.7 25.8

[0059] To confirm that these yeast homologs of the AN polypeptides areessential for survival of yeast, the gene encoding each of the homologswas, separately, deleted from the S. cerevisiae genome. To this end,standard methods for making yeast “knock outs” were used, as describedby Baudin et al., Nucl. Acids. Res. 21:3329-3330, 1993. Briefly, aportion of the yeast genome was amplified in a polymerase chain reaction(PCR) that employed two primers. The primers for L8004.2 were5′AGGAAAGTAGCTATCGTAACGGGTACTAATAGTAA (SEQ ID NO: 16) andTCTTGGTCTCTTGGCCTCCTCTAG 3′ 5′TACGCAGAGATATATTAAA (SEQ ID NO: 17).TGGGGGTTCTAGTTTCAACAATTTCGTTCAGAATGACACG 3′ The primers for D9798.4 were5′TTAACAGCCGCGCCCATCATGCAAGATCCTGATGGTATTGACA (SEQ ID NO: 18) andTTCTCTTGGCCTCCTCTAG 3′ 5′GCATATCAATTTTAACAGACCTCGCTG (SEQ ID NO: 19).AAAGACTCTGAATCCTCGTTCAGAATGACACG 3′

[0060] These primers hybridized to a portion of the 5′ and 3′ sequencesflanking the open reading frames of the yeast homologs and includenucleotides that are homologous to the HIS3 selectable marker. FollowingPCR amplification, the resulting crude mix was directly used totransform yeast, following a standard protocol.

[0061] Identification of AN97, AN17, AN80, and AN85 Genes in AdditionalAspergillus Strains

[0062] Now that the AN97, AN80, AN17, and AN85 genes and their yeasthomologs, L8004.2, YGR010W, L8543.16, and D9798.4, have been identifiedas essential for survival (as described below under “Examples”), thesegenes, or fragments thereof, can be used to detect homologous essentialgenes in other organisms. In particular, these genes can be used toanalyze various pathogenic and non-pathogenic strains of Aspergillus(e.g., Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger)and yeast (e.g., Candida albicans). In particular, fragments of anucleic acid (DNA or RNA) encoding an AN polypeptide or yeast homolog(or sequences complementary thereto) can be used as probes inconventional nucleic acid hybridization assays of pathogenic organisms(e.g., pathogenic Aspergillus strains). For example, nucleic acid probes(which typically are 8-30, or usually 15-20, nucleotides in length) canbe used to detect the AN97, AN17, AN80, AN85 genes or homologs thereofin art-known molecular biology methods, such as Southern blotting,Northern blotting, dot or slot blotting, PCR amplification methods,colony hybridization methods, and the like. Typically, anoligonucleotide probe based on the nucleic acid sequences describedherein, or fragments thereof, is labeled and used to screen a genomiclibrary or a cDNA library constructed from mRNA obtained from anAspergillus or yeast strain of interest. A suitable method of labelinginvolves using polynucleotide kinase to add ³²P-labeled ATP to theoligonucleotide used as the probe. This method is well known in the art,as are several other suitable methods (e.g., biotinylation and enzymelabeling).

[0063] Hybridization of the oligonucleotide probe to the cDNA library,or other nucleic acid sample, typically is performed under moderate tohigh stringency conditions. Nucleic acid duplex or hybrid stability isexpressed as the melting temperature or T_(m), which is the temperatureat which a probe dissociates from a target DNA. This melting temperatureis used to define the required stringency conditions. If sequences areto be identified that are related and substantially identical to theprobe, rather than identical, then it is useful to first establish thelowest temperature at which only homologous hybridization occurs with aparticular concentration of salt (e.g., SSC or SSPE). Then, assumingthat 1% mismatching results in a 1° C. decrease in the T_(m), thetemperature of the final wash in the hybridization reaction is reducedaccordingly (for example, if sequences having ≧95% identity with theprobe are sought, the final wash temperature is decreased by 5° C.). Inpractice, the change in T_(m) can be between 0.5° and 1.5° C. per 1%mismatch.

[0064] As used herein, high stringency conditions include, for example,hybridizing at 68° C. in 5× SSC/5× Denhardt's solution/1.0% SDS, or in0.5 M NaHPO₄ (pH 7.2)/1 mM EDTA/7% SDS, or in 50% formamide/0.25 MNaHPO₄ (pH 7.2)/0.25 M NaCl/1 mM EDTA/7% SDS; and washing in 0.2×SSC/0.1% SDS at room temperature or at 42° C., or in 0.1× SSC/0.1% SDSat 68° C., or in 40 mM NaHPO₄ (pH 7.2)/1 mM EDTA/5% SDS at 50° C., or in40 mM NaHPO₄ (pH 7.2) 1 mM EDTA/1% SDS at 50° C. Moderately stringentconditions include washing in 3× SSC at 42° C. The parameters of saltconcentration and temperature can be varied to achieve the optimal levelof identity between the probe and the target nucleic acid. Additionalguidance regarding such conditions is readily available in the art, forexample, by Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995,Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.) atUnit 2.10.

[0065] In one approach, cDNA libraries constructed from pathogenic ornon-pathogenic Aspergillus or yeast strains can be screened. Forexample, such strains can be screened for AN97, AN17, AN85, or AN80expression by Northern blot analysis. Upon detection of AN97, AN17,AN85, or AN80 transcripts or transcripts of homologs thereof, cDNAlibraries can be constructed from RNA isolated from the appropriatestrain, utilizing standard techniques well known to those of skill inthe art. Alternatively, a total genomic DNA library can be screenedusing an AN97, AN17, AN85, or AN80 probe (or a probe directed to ahomolog thereof).

[0066] New gene sequences can be isolated, for example, by performingPCR using two degenerate oligonucleotide primer pools designed on thebasis of nucleotide sequences within the AN97, AN17, AN85 or AN80 genes,or their homologs, as depicted herein. The template for the reaction canbe cDNA obtained by reverse transcription of mRNA prepared from strainsknown or suspected to express an AN97, AN17, AN85, or AN80 allele or anallele of a homolog thereof. The PCR product can be subcloned andsequenced to ensure that the amplified sequences represent the sequencesof a new AN97, AN17, AN85, or AN80 nucleic acid sequence, or a sequenceof a homolog thereof.

[0067] The PCR fragment can then be used to isolate a full length cDNAclone by a variety of known methods. For example, the amplified fragmentcan be labeled and used to screen a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to screen a genomiclibrary.

[0068] PCR technology also can be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid can then be “tailed” (e.g., with guanines) using astandard terminal transferase reaction, the hybrid can be digested withRNase H, and second strand synthesis can then be primed (e.g., with apoly-C primer). Thus, cDNA sequences upstream of the amplified fragmentcan easily be isolated. For a review of useful cloning strategies, seee.g., Sambrook et al., supra; and Ausubel et al., supra.

[0069] Now that the AN97, AN17, AN85, and AN80 genes and their homologshave been cloned, synthesis of the AN polypeptides or their homologs (oran antigenic fragment thereof) for use as antigens, or for otherpurposes, can readily be accomplished using any of the various art-knowntechniques. For example, an AN polypeptide or homolog, or an antigenicfragment(s), can be synthesized chemically in vitro, or enzymatically(e.g., by in vitro transcription and translation). Alternatively, thegene can be expressed in, and the polypeptide purified from, a cell(e.g., a cultured cell) by using any of the numerous, available geneexpression systems. For example, the polypeptide antigen can be producedin a prokaryotic host (e.g., E. coli or B. subtilis) or in eukaryoticcells, such as yeast cells or insect cells (e.g., by using abaculovirus-based expression vector).

[0070] Proteins and polypeptides can also be produced in plant cells, ifdesired. For plant cells viral expression vectors (e.g., cauliflowermosaic virus and tobacco mosaic virus) and plasmid expression vectors(e.g., Ti plasmid) are suitable. Such cells are available from a widerange of sources (e.g., the American Type Culture Collection, Rockland,Md.; also, see, e.g., Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1994). The optimal methods oftransformation or transfection and the choice of expression vehicle willdepend on the host system selected. Transformation and transfectionmethods are described, e.g., in Ausubel et al., supra; expressionvehicles may be chosen from those provided, e.g., in Cloning Vectors: ALaboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987). The hostcells harboring the expression vehicle can be cultured in conventionalnutrient media, adapted as needed for activation of a chosen gene,repression of a chosen gene, selection of transformants, oramplification of a chosen gene.

[0071] If desired, AN polypeptides or their homologs can be produced asfusion proteins. For example, the expression vector pUR278 (Ruther etal., EMBO J., 2:1791, 1983) can be used to create lacZ fusion proteins.The art-known pGEX vectors can be used to express foreign polypeptidesas fusion proteins with glutathione S-transferase (GST). In general,such fusion proteins are soluble and can be easily purified from lysedcells by adsorption to glutathione-agarose beads followed by elution inthe presence of free glutathione. The pGEX vectors are designed toinclude thrombin or factor Xa protease cleavage sites so that the clonedtarget gene product can be released from the GST moiety.

[0072] In an exemplary insect cell expression system, a baculovirus suchas Autographa califormica nuclear polyhedrosis virus (AcNPV), whichgrows in Spodoptera frugiperda cells, can be used as a vector to expressforeign genes. A coding sequence encoding an AN polypeptide or homologcan be cloned into a non-essential region (for example the polyhedringene) of the viral genome and placed under control of a promoter, e.g.,the polyhedrin promoter or an exogenous promoter. Successful insertionof a gene encoding an AN polypeptide or homolog can result ininactivation of the polyhedrin gene and production of non-occludedrecombinant virus (i.e., virus lacking the proteinaceous coat encoded bythe polyhedrin gene). These recombinant viruses are then used to infectinsect cells (e.g., Spodoptera frugiperda cells) in which the insertedgene is expressed (see, e.g., Smith et al., J. Virol., 46:584, 1983;Smith, U.S. Pat. No. 4,215,051).

[0073] In mammalian host cells, a number of viral-based expressionsystems can be utilized. When an adenovirus is used as an expressionvector, the nucleic acid sequence encoding the AN polypeptide or homologcan be ligated to an adenovirus transcription/translation controlcomplex, e.g., the late promoter and tripartite leader sequence. Thischimeric gene can then be inserted into the adenovirus genome by invitro or in vivo recombination. Insertion into a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing an AN97, AN17, AN85, orAN80 gene product in infected hosts (see, e.g., Logan, Proc. Natl. Acad.Sci. USA, 81:3655, 1984).

[0074] Specific initiation signals may be required for efficienttranslation of inserted nucleic acid sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire native gene (e.g., AN97) or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. In other cases, exogenous translational control signals,including, perhaps, the ATG initiation codon, should be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entiresequence. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, or transcription terminators (Bittneret al., Methods in Enzymol., 153:516, 1987).

[0075] The AN polypeptides and homologs can be expressed individually oras fusions with a heterologous polypeptide, such as a signal sequence orother polypeptide having a specific cleavage site at the N-and/orC-terminus of the protein or polypeptide. The heterologous signalsequence selected should be one that is recognized and processed, i.e.,cleaved by a signal peptidase, by the host cell in which the fusionprotein is expressed.

[0076] A host cell can be chosen that modulates the expression of theinserted sequences, or modifies and processes the gene product in aspecific, desired fashion. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products may facilitate optimalfunctioning of the protein. Various host cells have characteristic andspecific mechanisms for post-translational processing and modificationof proteins and gene products. Appropriate cell lines or host systemsfamiliar to those of skill in the art of molecular biology can be chosento ensure the correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells that possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product can be used. Suchmammalian host cells include, but are not limited to, CHO, VERO, BHK,HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.

[0077] If desired, the AN polypeptide or homolog thereof can be producedby a stably-transfected mammalian cell line. A number of vectorssuitable for stable transection of mammalian cells are available to thepublic, see, e.g., Pouwels et al. (supra); methods for constructing suchcell lines are also publicly known, e.g., in Ausubel et al. (supra). Inone example, cDNA encoding the protein is cloned into an expressionvector that includes the dihydrofolate reductase (DHFR) gene.Integration of the plasmid and, therefore, the AN polypeptide-encodinggene into the host cell chromosome is selected for by including 0.01-300μM methotrexate in the cell culture medium (as described in Ausubel etal., supra). This dominant selection can be accomplished in most celltypes.

[0078] Recombinant protein expression can be increased by DHFR-mediatedamplification of the transfected gene. Methods for selecting cell linesbearing gene amplifications are described in Ausubel et al. (supra);such methods generally involve extended culture in medium containinggradually increasing levels of methotrexate. DHFR-containing expressionvectors commonly used for this purpose include pCVSEII-DHFR andpAdD26SV(A) (described in Ausubel et al., supra).

[0079] A number of other selection systems can be used, including butnot limited to the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyl-transferase, and adeninephosphoribosyltransferase genes can be employed in tk, hgprt, or aprtcells, respectively. In addition, gpt, which confers resistance tomycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA, 78:2072,1981); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol., 150:1, 1981); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene, 30:147, 1981),can be used.

[0080] Alternatively, any fusion protein can be readily purified byutilizing an antibody or other molecule that specifically binds thefusion protein being expressed. For example, a system described inJanknecht et al., Proc. Natl. Acad. Sci. USA, 88:8972 (1981), allows forthe ready purification of non-denatured fusion proteins expressed inhuman cell lines. In this system, the gene of interest is subcloned intoa vaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns,and histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0081] Alternatively, an AN polypeptide or homolog, or a portionthereof, can be fused to an immunoglobulin Fc domain. Such a fusionprotein can be readily purified using a protein A column, for example.Moreover, such fusion proteins permit the production of a chimeric formof an AN polypeptide or homolog having increased stability in vivo.

[0082] Once the recombinant AN polypeptide (or homolog) is expressed, itcan be isolated (i.e., purified). Secreted forms of the polypeptides canbe isolated from cell culture media, while non-secreted forms must beisolated from the host cells. Polypeptides can be isolated by affinitychromatography. For example, an anti-AN97 antibody (e.g., produced asdescribed herein) can be attached to a column and used to isolate theprotein. Lysis and fractionation of cells harboring the protein prior toaffinity chromatography can be performed by standard methods (see, e.g.,Ausubel et al., supra). Alternatively, a fusion protein can beconstructed and used to isolate an AN polypeptide (e.g., an AN97-maltosebinding fusion protein, an AN97-β-galactosidase fusion protein, or anAN97-trpE fusion protein; see, e.g., Ausubel et al., supra; New EnglandBiolabs Catalog, Beverly, Mass.). The recombinant protein can, ifdesired, be further purified, e.g., by high performance liquidchromatography using standard techniques (see, e.g., Fisher, LaboratoryTechniques In Biochemistry And Molecular Biology, eds., Work and Burdon,Elsevier, 1980).

[0083] Given the amino acid sequences described herein, polypeptidesuseful in practicing the invention, particularly fragments of AN97,AN17, AN85, AN80 from pathogenic Aspergillus strains, and fragments ofD9798.4, L8004.2, L8543.16, and YGR010W from yeast, can be produced bystandard chemical synthesis (e.g., by the methods described in SolidPhase Peptide Synthesis, 2nd ed., The Pierce Chemical Co., Rockford,Ill., 1984) and used as antigens, for example.

[0084] Antibodies

[0085] AN97, AN17, AN85, or AN80 polypeptides (or antigenic fragments oranalogs of such polypeptide) can be used to raise antibodies useful inthe invention, and such polypeptides can be produced by recombinant orpeptide synthetic techniques (see, e.g., Solid Phase Peptide Synthesis,supra; Ausubel et al., supra). Likewise, antibodies can be raisedagainst the yeast homologs. In general, the polypeptides can be coupledto a carrier protein, such as KLH, as described in Ausubel et al.,supra, mixed with an adjuvant, and injected into a host mammal.Antibodies can be purified, for example, by affinity chromatographymethods in which the polypeptide antigen is immobilized on a resin.

[0086] In particular, various host animals can be immunized by injectionof a polypeptide of interest. Examples of suitable host animals includerabbits, mice, guinea pigs, and rats. Various adjuvants can be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete),adjuvant mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG (bacilleCalmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies areheterogeneous populations of antibody molecules derived from the sera ofthe immunized animals.

[0087] Antibodies within the invention include monoclonal antibodies,polyclonal antibodies, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, and molecules producedusing a Fab expression library.

[0088] Monoclonal antibodies (mAbs), which are homogeneous populationsof antibodies to a particular antigen, can be prepared using the ANpolypeptides or homologs thereof and standard hybridoma technology (see,e.g., Kohler et al., Nature, 256:495, 1975; Kohler et al., Eur. J.Immunol., 6:511, 1976; Kohler et al., Eur. J. Immunol., 6:292, 1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y., 1981; Ausubel et al., supra).

[0089] In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture, such as those described in Kohler etal., Nature, 256:495, 1975, and U.S. Pat. No. 4,376,110; the humanB-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72, 1983;Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026, 1983); and theEBV-hybridoma technique (Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96, 1983). Such antibodies can be ofany immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and anysubclass thereof. The hybridomas producing the mAbs of this inventioncan be cultivated in vitro or in vivo.

[0090] Once produced, polyclonal or monoclonal antibodies are tested forspecific recognition of an AN polypeptide or homolog thereof in animmunoassay, such as a Western blot or immunoprecipitation analysisusing standard techniques, e.g., as described in Ausubel et al., supra.Antibodies that specifically bind to AN97, AN17, AN85, or AN80, orconservative variants and homologs thereof, are useful in the invention.For example, such antibodies can be used in an immunoassay to detectAN97 in pathogenic or non-pathogenic strains of Aspergillus (e.g., inAspergillus extracts).

[0091] Preferably, antibodies of the invention are produced usingfragments of the AN polypeptides that appear likely to be antigenic, bycriteria such as high frequency of charged residues. In one specificexample, such fragments are generated by standard techniques of PCR, andare then cloned into the pGEX expression vector (Ausubel et al., supra).Fusion proteins are expressed in E. coli and purified using aglutathione agarose affinity matrix as described in Ausubel, et al.,supra.

[0092] If desired, several (e.g., two or three) fusions can be generatedfor each protein, and each fusion can be injected into at least tworabbits. Antisera can be raised by injections in a series, typicallyincluding at least three booster injections. Typically, the antisera ischecked for its ability to immunoprecipitate a recombinant ANpolypeptide or homolog, or unrelated control proteins, such asglucocorticoid receptor, chloramphenicol acetyltransferase, orluciferase.

[0093] Techniques developed for the production of “chimeric antibodies”(Morrison et al., Proc. Natl. Acad. Sci., 81:6851, 1984; Neuberger etal., Nature, 312:604, 1984; Takeda et al., Nature, 314:452, 1984) can beused to splice the genes from a mouse antibody molecule of appropriateantigen specificity together with genes from a human antibody moleculeof appropriate biological activity. A chimeric antibody is a molecule inwhich different portions are derived from different animal species, suchas those having a variable region derived from a murine mAb and a humanimmunoglobulin constant region.

[0094] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. Nos. 4,946,778; and 4,946,778 and 4,704,692)can be adapted to produce single chain antibodies against an ANpolypeptide or homolog. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

[0095] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragments caninclude but are not limited to F(ab′)₂ fragments, which can be producedby pepsin digestion of the antibody molecule, and Fab fragments, whichcan be generated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0096] Polyclonal and monoclonal antibodies that specifically bind ANpolypeptides or homologs can be used, for example, to detect expressionof an AN97, AN17, AN85, AN80 gene or homolog in another strain ofAspergillus. For example, AN97 polypeptide can be readily detected inconventional immunoassays of Aspergillus cells or extracts. Examples ofsuitable assays include, without limitation, Western blotting, ELISAs,radioimmune assays, and the like.

[0097] Assay for Antifungal Agents

[0098] The invention provides a method for identifying an antifungalagent(s). Although the inventors are not bound by any particular theoryas to the biological mechanism involved, the new antifungal agents arethought to inhibit specifically the function of the AN polypeptides orexpression of the AN97, AN17, AN85, or AN80 genes, or homologs thereof.Screening for antifungal agents can be rapidly accomplished byidentifying those compounds (e.g., polypeptides, ribonucleic acids(including ribozymes), nucleic acids (including antisense nucleicacids), or small molecules) that specifically bind to an AN polypeptide.A homolog of an AN polypeptide (e.g., D9798.4, L8004.2, L8543.16, orYGR010W) can be substituted for the AN polypeptide in the methodssummarized herein. Specific binding of a test compound to an ANpolypeptide can be detected, for example, in vitro by reversibly orirreversibly immobilizing the test compound(s) on a substrate, e.g., thesurface of a well of a 96-well polystyrene microtiter plate. Methods forimmobilizing polypeptides and other small molecules are well known inthe art. For example, the microtiter plates can be coated with an ANpolypeptide (or a combination of AN polypeptides and/or homologs) byadding the polypeptide(s) in a solution (typically, at a concentrationof 0.05 to 1 mg/ml in a volume of 1-100 μl) to each well, and incubatingthe plates at room temperature to 37° C. for 0.1 to 36 hours.Polypeptides that are not bound to the plate can be removed by shakingthe excess solution from the plate, and then washing the plate (once orrepeatedly) with water or a buffer. Typically, the AN polypeptide orhomolog is contained in water or a buffer. The plate is then washed witha buffer that lacks the bound polypeptide. To block the freeprotein-binding sites on the plates, the plates are blocked with aprotein that is unrelated to the bound polypeptide. For example, 300 μlof bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HClis suitable. Suitable substrates include those substrates that contain adefined cross-linking chemistry (e.g., plastic substrates, such aspolystyrene, styrene, or polypropylene substrates from Corning CostarCorp. (Cambridge, Mass.), for example). If desired, a beaded particle,e.g., beaded agarose or beaded sepharose, can be used as the substrate.

[0099] Binding of the test compound to the new AN polypeptides (orhomologs thereof) can be detected by any of a variety of art-knownmethods. For example, an antibody that specifically binds an ANpolypeptide can be used in an immunoassay. If desired, the antibody canbe labeled (e.g., fluorescently or with a radioisotope) and detecteddirectly (see, e.g., West and McMahon, J. Cell Biol. 74:264, 1977).Alternatively, a second antibody can be used for detection (e.g., alabeled antibody that binds the Fc portion of an anti-AN97 antibody). Inan alternative detection method, the AN polypeptide is labeled, and thelabel is detected (e.g., by labeling an AN polypeptide with aradioisotope, fluorophore, chromophore, or the like). In still anothermethod, the AN polypeptide is produced as a fusion protein with aprotein that can be detected optically, e.g., green fluorescent protein(which can be detected under UV light). In an alternative method, the ANpolypeptide can be produced as a fusion protein with an enzyme having adetectable enzymatic activity, such as horse radish peroxidase, alkalinephosphatase, α-galactosidase, or glucose oxidase. Genes encoding all ofthese enzymes have been cloned and are readily available for use bythose of skill in the art. If desired, the fusion protein can include anantigen, and such an antigen can be detected and measured with apolyclonal or monoclonal antibody using conventional methods. Suitableantigens include enzymes (e.g., horse radish peroxidase, alkalinephosphatase, and α-galactosidase) and non-enzymatic polypeptides (e.g.,serum proteins, such as BSA and globulins, and milk proteins, such ascaseins).

[0100] In various in vivo methods for identifying polypeptides that bindAN polypeptides, the conventional two-hybrid assays of protein/proteininteractions can be used (see e.g., Chien et al., Proc. Natl. Acad. Sci.USA, 88:9578, 1991; Fields et al., U.S. Pat. No. 5,283,173; Fields andSong, Nature, 340:245, 1989; Le Douarin et al., Nucleic Acids Research,23:876, 1995; Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10315-10320,1996; and White, Proc. Natl. Acad. Sci. USA, 93:10001-10003, 1996). Kitsfor practicing various two-hybrid methods are commercially available(e.g., from Clontech; Palo Alto, Calif.).

[0101] Generally, the two-hybrid methods involve in vivo reconstitutionof two separable domains of a transcription factor. The DNA bindingdomain (DB) of the transcription factor is required for recognition of achosen promoter. The activation domain (AD) is required for contactingother components of the host cell's transcriptional machinery. Thetranscription factor is reconstituted through the use of hybridproteins. One hybrid is composed of the AD and a first protein ofinterest. The second hybrid is composed of the DB and a second proteinof interest. In cases where the first and second proteins of interestinteract with each other, the AD and DB are brought into close physicalproximity, thereby reconstituting the transcription factor. Associationof the proteins can be measured by assaying the ability of thereconstituted transcription factor to activate transcription of areporter gene.

[0102] Useful reporter genes are those that are operably linked to apromoter which is specifically recognized by the DB. Typically, thetwo-hybrid system employs the yeast Saccharomyces cerevisiae andreporter genes, the expression of which can be selected underappropriate conditions. Other eukaryotic cells, including mammalian andinsect cells, can be used, if desired. The two-hybrid system provides aconvenient method for cloning a gene encoding a polypeptide (i.e., acandidate antifungal agent) that binds a second, preselected polypeptide(e.g., AN97). Typically, though not necessarily, a cDNA library isconstructed such that randomly generated sequences are fused to the AD,and the protein of interest (e.g., AN97 or AN80) is fused to the DB.

[0103] In such two-hybrid methods, two fusion proteins are produced. Onefusion protein contains the AN polypeptide (or homolog thereof) fused toeither a transactivator domain or DNA binding domain of a transcriptionfactor (e.g., of Ga14). The other fusion protein contains a testpolypeptide fused to either the DNA binding domain or a transactivatordomain of a transcription factor. Once brought together in a single cell(e.g., a yeast cell or mammalian cell), one of the fusion proteinscontains the transactivator domain and the other fusion protein containsthe DNA binding domain. Therefore, binding of the AN polypeptide to thetest polypeptide (i.e., candidate antifungal agent) reconstitutes thetranscription factor. Reconstitution of the transcription factor can bedetected by detecting expression of a gene (i.e., a reporter gene) thatis operably linked to a DNA sequence that is bound by the DNA bindingdomain of the transcription factor.

[0104] The methods described above can be used for high throughputscreening of numerous test compounds to identify candidate antifungal(or anti-yeast) agents. Having identified a test compound as a candidateantifungal agent, the candidate antifungal agent can be further testedfor inhibition of fungal growth in vitro or in vivo (e.g., using ananimal, e.g., rodent, model system) if desired. Using other, art-knownvariations of such methods, one can test the ability of a nucleic acid(e.g., DNA or RNA) used as the test compound to bind an AN polypeptideor homolog thereof.

[0105] In vitro, further testing can be accomplished by means known tothose in the art such as an enzyme inhibition assay or a whole-cellfungal growth inhibition assay. For example, an agar dilution assayidentifies a substance that inhibits fungal growth. Microtiter platesare prepared with serial dilutions of the test compound; adding to thepreparation a given amount of growth substrate; and providing apreparation of Aspergillus spores. Inhibition of growth is determined,for example, by observing changes in optical densities of the fungalcultures.

[0106] Inhibition of fungal growth is demonstrated, for example, bycomparing (in the presence and absence of a test compound) the rate ofgrowth or the absolute growth of fungal sporulation or nuclei.Inhibition includes a reduction of one of the above measurements by atleast 20% (e.g., at least 25%, 30%, 40%, 50%, 75%, 80%, or 90%).

[0107] Rodent (e.g., murine) and bovine animal models of aspergillosisare known to those of skill in the art, and such animal model systemsare accepted for screening antifungal agents as an indication of theirtherapeutic efficacy in human patients (Rhodes et al., J. Med. and Vet.Myco., 30:51-57, 1992). Indeed, the clinical manifestations of bovineaspergillosis show many pathological similarities to aspergillosis inhumans and rodents. In a typical in vivo assay, an animal is infectedwith a pathogenic Aspergillus strain, e.g., by inhalation of Aspergillusspores (i.e., conidia), and conventional methods and criteria are usedto diagnose the mammal as being afflicted with aspergillosis. Thecandidate antifungal agent then is administered to the mammal at adosage of 1-100 mg/kg of body weight, and the mammal is monitored forsigns of amelioration of disease. Alternatively, the test compound canbe administered to the mammal prior to infecting the mammal withAspergillus, and the ability of the treated mammal to resist infectionis measured. Of course, the results obtained in the presence of the testcompound are compared with results in control animals, which are nottreated with the test compound. Administration of candidate antifungalagent to the mammal can be carried out as described below, for example.

[0108] Antisense Methods

[0109] Antisense approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to AN97, AN17, AN80, or AN85mRNA. The antisense oligonucleotides bind to the AN97, AN17, AN80, orAN85 coding sequences and/or mRNA transcripts and inhibit transcriptionand/or translation. Absolute complementarity is not required. A sequence“complementary” to a portion of an RNA, as referred to herein, means asequence having sufficient complementarity to be able to hybridize withthe RNA and form a stable duplex; in the case of double-strandedantisense nucleic acids, a single strand of the duplex DNA can betested, or triplex formation can be assayed. The ability to hybridizewill depend on both the degree of complementarity and the length of theantisense nucleic acid. Generally, the longer the hybridizing nucleicacid, the more base mismatches with an RNA it may contain and still forma stable duplex (or triplex, as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0110] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well (Wagner, Nature, 372:333, 1984). Thus,oligonucleotides complementary to either the 5′- or 3′- non-translated,non-coding regions of the AN97, AN17, AN80, or AN85 genes, or theiryeast homologs D9798.4, L8543.16, YGR010W, of L8004.2, as represented bySEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16 can be used in an antisenseapproach to inhibit translation of the endogenous sequences.Oligonucleotides complementary to the 5′ untranslated region of the mRNAtypically also include the complement of the AUG start codon.

[0111] Antisense oligonucleotides complementary to mRNA coding regionsare less preferred inhibitors of translation, but can be used inaccordance with the invention. Whether designed to hybridize to the 5′-,3′-, or coding region of the mRNA, antisense nucleic acids should be atleast six nucleotides in length (e.g., oligonucleotides ranging from 6to about 50 nucleotides in length). In specific aspects, theoligonucleotide is at least 10 nucleotides, at least 15 nucleotides, orat least 25 nucleotides.

[0112] Regardless of the choice of target sequence, in vitro studiestypically are first performed to quantitate the ability of the antisenseoligonucleotide to inhibit gene expression. Typically, these studiesutilize controls that distinguish between antisense gene inhibition andnonspecific biological effects of oligonucleotides. Generally, thesestudies compare levels of the target RNA or protein with that of aninternal control RNA or protein. Additionally, it is envisioned thatresults obtained using the antisense oligonucleotide are compared withthose obtained using a control oligonucleotide. Typically, the controloligonucleotide is of approximately the same length as the testoligonucleotide and that the nucleotide sequence of the oligonucleotidediffers from the antisense sequence no more than is necessary to preventspecific hybridization to the target sequence.

[0113] The antisense oligonucleotides can be DNA or RNA, or chimericmixtures, or derivatives or modified versions thereof, and can besingle-stranded or double-stranded. The oligonucleotides can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (as described, e.g., inLetsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553, 1989; Lemaitre etal., Proc. Natl. Acad. Sci. USA, 84:648, 1987; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134), or hybridization-triggered cleavage agents (see, e.g., Krolet al., BioTechniques, 6:958, 1988), or intercalating agents (see, e.g.,Zon, Pharm. Res., 5:539, 1988). To this end, the oligonucleotide can beconjugated to another molecule, e.g., a peptide, hybridization triggeredcross-linking agent, transport agent, or hybridization-triggeredcleavage agent.

[0114] The antisense oligonucleotide can include at least one modifiedbase moiety selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 2-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0115] The antisense oligonucleotide can also include at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0116] In yet another embodiment, the antisense oligonucleotide includesat least one modified phosphate backbone, e.g., a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphorodiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal, or an analog of any of these backbones.

[0117] In addition, the antisense oligonucleotide can be an α-anomericoligonucleotide that forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gautier et al., Nucl. Acids. Res., 15:6625,1987). The oligonucleotide can be a 2′-0-methylribonucleotide (Inoue etal., Nucl. Acids Res., 15:6131, 1987), or a chimeric RNA-DNA analog(Inoue et al., FEBS Lett., 215:327, 1987).

[0118] Antisense oligonucleotides of the invention can be synthesized bystandard methods known in the art, e.g., by use of an automated DNAsynthesizer (such as are commercially available from Biosearch, AppliedBiosystems, etc.). As examples, phosphorothioate oligonucleotides can besynthesized by the method of Stein et al., Nucl. Acids Res., 16:3209,1988, and methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin et al., Proc. Natl. Acad.Sci. USA, 85:7448, 1988).

[0119] While antisense nucleotides complementary to the AN97, AN17,AN80, AN85, D9798.4, L8543.16, YGR010W, or L8004.2 coding regionsequence could be used, those complementary to the transcribeduntranslated region are preferred. Generally, such antisenseoligonucleotides are 10-100 nucleotides in length (e.g., 15-50nucleotides). Pathogenic microorganisms, such as Aspergillus, canspontaneously phagocytose oligonucleotides. Accordingly, these antisenseoligonucleotides can be administered systemically or locally to apatient suffering from a pathogen infection in order to deliver theantisense oligonucleotides to the infectious organism in a method oftreatment. For example, such antisense oligonucleotides can be used toinhibit expression of an AN polypeptide and thereby treat or inhibitfungal infections. A suitable approach uses a recombinant DNA constructin which the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect fungal cells in the patient will result in the transcriptionof sufficient amounts of single stranded nucleic acids that formcomplementary base pairs with the endogenous transcripts encoding ANpolypeptides and thereby prevent translation of the mRNA. For example, avector can be introduced in vivo such that it is taken up by a cell anddirects the transcription of an antisense RNA. Such a vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA.

[0120] Appropriate vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression infungal cells. Expression of the sequence encoding the antisense RNA canbe by any promoter known in the art to act in fungi, e.g. Aspergillus,cells. Such promoters can be inducible or constitutive, such as analcohol dehydrogenase promoter (e.g., alcA) and a nitrate reductasepromoter (e.g., niiA). Any type of plasmid, cosmid, or viral vector canbe used to prepare the recombinant DNA construct which can beadministered systemically or directly to the infected tissue.

[0121] Ribozymes

[0122] Ribozyme molecules designed to catalytically cleave mRNAtranscripts encoding AN polypeptides also can be used to preventtranslation of mRNA and expression of the AN polypeptides (see, e.g.,PCT Publication WO 90/11364; Saraver et al., Science, 247:1222, 1990).Various ribozymes that cleave mRNA at site-specific recognitionsequences can be used to destroy mRNAs encoding the AN polypeptides(e.g., the use of hammerhead ribozymes). Hammerhead ribozymes cleavemRNAs at locations dictated by flanking regions that form complementarybase pairs with the target mRNA. It is recommended that the target mRNAhave the following sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is known in the art (Haseloff et al.,Nature, 334:585, 1988). There are numerous examples of potentialhammerhead ribozyme cleavage sites within the nucleotide sequence ofcDNAs encoding AN polypeptides (FIGS. 1 to 3). Typically, the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the mRNA encoding the AN polypeptide in order to increaseefficiency and minimize the intracellular accumulation of non-functionalmRNA transcripts.

[0123] The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”), such as the onethat occurs naturally in Tetrahymena Thermophila (known as the IVS orL-19 IVS RNA), and which has been extensively described by Cech and hiscollaborators (Zaug et al., Science, 224:574, 1984; Zaug et al.,Science, 231:470, 1986; Zug et al., Nature, 324:429, 1986; PCTApplication No. WO 88/04300; and Been et al., Cell, 47:207, 1986). TheCech-type ribozymes have an eight base-pair sequence that hybridizes toa target RNA sequence, whereafter cleavage of the target RNA takesplace. The invention encompasses those Cech-type ribozymes that targeteight base-pair active site sequences present in AN polypeptides.

[0124] As in the antisense approach, the ribozymes can be composed ofmodified oligonucleotides (e.g., for improved stability, targeting,etc.), and should be delivered to cells that express the AN polypeptide.A typical method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive promoter, e.g.,a pol III or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous mRNAsencoding AN polypeptides and inhibit translation thereof. Becauseribozymes, unlike typical antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

[0125] Pharmaceutical Formulations

[0126] Treatment includes administering a pharmaceutically effectiveamount of a composition containing an antifungal agent to a subject inneed of such treatment, thereby inhibiting fungal growth in the subject.Such a composition typically contains from about 0.1 to 90% by weight(such as 1 to 20% or 1 to 10%) of an antifungal agent of the inventionin a pharmaceutically acceptable carrier.

[0127] Solid formulations of the compositions for oral administrationmay contain suitable carriers or excipients, such as corn starch,gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin,mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, oralginic acid. Disintegrators that can be used include, withoutlimitation, micro-crystalline cellulose, corn starch, sodium starchglycolate and alginic acid. Tablet binders that may be used includeacacia, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose,starch, and ethylcellulose. Lubricants that may be used includemagnesium stearates, stearic acid, silicone fluid, talc, waxes, oils,and colloidal silica.

[0128] Liquid formulations of the compositions for oral administrationprepared in water or other aqueous vehicles may contain varioussuspending agents such as methylcellulose, alginates, tragacanth,pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinylalcohol. The liquid formulations may also include solutions, emulsions,syrups and elixirs containing, together with the active compound(s),wetting agents, sweeteners, and coloring and flavoring agents. Variousliquid and powder formulations can be prepared by conventional methodsfor inhalation into the lungs of the mammal to be treated.

[0129] Injectable formulations of the compositions may contain variouscarriers such as vegetable oils, dimethylacetamide, dimethylformamide,ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).For intravenous injections, water soluble versions of the compounds maybe administered by the drip method, whereby a pharmaceutical formulationcontaining the antifungal agent and a physiologically acceptableexcipient is infused. Physiologically acceptable excipients may include,for example, 5% dextrose, 0.9% saline, Ringer's solution or othersuitable excipients. Intramuscular preparations, a sterile formulationof a suitable soluble salt form of the compounds can be dissolved andadministered in a pharmaceutical excipient such as Water-for-Injection,0.9% saline, or 5% glucose solution. A suitable insoluble form of thecompound may be prepared and administered as a suspension in an aqueousbase or a pharmaceutically acceptable oil base, such as an ester of along chain fatty acid, (e.g., ethyl oleate).

[0130] A topical semi-solid ointment formulation typically contains aconcentration of the active ingredient from about 1 to 20%, e.g., 5 to10% in a carrier such as a pharmaceutical cream base. Variousformulations for topical use include drops, tinctures, lotions, creams,solutions, and ointments containing the active ingredient and varioussupports and vehicles.

[0131] The optimal percentage of the antifungal agent in eachpharmaceutical formulation varies according to the formulation itselfand the therapeutic effect desired in the specific pathologies andcorrelated therapeutic regimens. Appropriate dosages of the antifungalagents can readily be determined by those of ordinary skill in the artof medicine by monitoring the mammal for signs of disease ameliorationor inhibition, and increasing or decreasing the dosage and/or frequencyof treatment as desired. The optimal amount of the antifungal compoundused for treatment of conditions caused by or contributed to by fungalinfection may depend upon the manner of administration, the age and thebody weight of the subject and the condition of the subject to betreated. Generally, the antifungal compound is administered at a dosageof 1 to 100 mg/kg of body weight, and typically at a dosage of 1 to 10mg/kg of body weight.

EXAMPLE

[0132] In this example, the identification and cloning of AN97, AN17,AN85, and AN80 are described.

[0133] A library of approximately 1,000 A. nidulans mutants wasobtained, which was prepared using 4-nitroquinoline as a mutagen, asdescribed previously (Harris et al., Genetics 136:517-532 1994). Toidentify strains having a temperature-sensitive mutation in an essentialgene, the collection of 1,000 strains was grown at the permissivetemperature of 28° C. for 16 hours in minimal medium (MN; pH 6.5, 1%glucose, nitrate salts and trace elements as described in Kafer, Adv.Genet. 19:33-131, 1977). The trace element solution was stored at 40 inthe dark; each liter contained 40 mg Na₂B₄O₇ (10 H₂O), 400 mg cupricsulfate (5 H₂O), 1 g ferric phosphate (4 H₂O), 600 mg manganese sulfate(4 H₂O ), 800 mg disodium molybdate (2 H₂O), and 8 g zinc sulfate (7H₂O). Slat solution was stored at 4° C. after adding 2 ml chloroform asa preservative; each liter contained 26 g potassium chloride, 26 gmagnesium sulfate (7 H₂O) 76 g monobasic potassium phosphate and 50 mLtrace element solution. Supplement solution was sterilized byautoclaving for 15 minutes and stored in a light-proof container due tothe reactivity of riboflavin. Each liter contains 100 mg nicotinic acid,250 mg riboflavin, 200 mg pantothenic acid, 50 mg pyridoxin, 1 mgbiotin, and 20 mg p-aminobenzoic acid.

[0134] Condidia (2×10⁶/ml in sterile, distilled water) were mutagenizedwith NQO (4 μg/ml) for 30 minutes at 37° C. with constant shaking.Diluting the conidia with an equal volume of 5% sodium thiosulfateinactivated the NQO. Mutagenized conidia were diluted and plated ontoCM+TRITON X-100 plates (from Union Carbide Chemicals,) and incubated at28° C. for 3 days. Colonies were replica plated and the replica platedplates were incubated at 28° C. and 42° C. Putativetemperature-sensitive mutants were picked and retested, then stored as acolony plug in 15% glycerol at −70° C.

[0135] The cells were replica plated and shifted to 42° C. for 24 hours.Strains that grew poorly or not at all were selected, because they weremost likely to represent strains having a mutation in an essential gene.After 1 round of subjecting the collection of cells to the temperatureshift, approximately 100 strains (10% of the strains) were identified ashaving failed to recover once they were shifted to the second permissivetemperature. These 100 strains were again grown at a first permissivetemperature, followed by 24 hours at 42° C., and 24 or 48 hours at 28°C. (the second permissive temperature). After this second round ofselection, 10 strains were identified as having failed to recover, andtherefore as containing a temperature sensitive mutation in an essentialgene.

[0136] Complementation analysis was used to identify the essential genecontaining the mutation for each strain. Each of the 10 mutant strainswas transformed, separately, with an Aspergillus genomic cosmid librarycontaining an ArgB marker in a pCosAx vector (Adams et al., FEMSMicrobiol. Lett., 122:227-231 1994). The strains were grown for 3-4 daysat 28° C., replica plated, and shifted to 42° C. for a maximum of 3days. Strains that grew were collected, and the cosmid DNA was packagedby “selfing” the organism to force it to undergo meiosis. In thismethod, a colony is picked and grown on a separate plate (whichtypically is sealed to prevent contamination). The resulting spores thenare picked and grown in liquid culture, prior to isolating the DNA. Thecosmid was packaged using GIGAPACK III Gold packaging system(Stratagene; La Jolla, Calif.), which produced plasmids that weresubsequently isolated, purified, and used to transform bacteria foramplification, isolation, purification, and sequencing.

[0137] In one of the resulting strains, the mutation was in a genedesignated “AN97,” indicating that in A. nidulans this gene is essentialfor survival. The amino acid sequence of the AN97 polypeptide and theAN97 gene of A. nidulans are provided in FIG. 1 as SEQ ID NOs:1 and 2,respectively.

[0138] In a second strain, the mutation was in a gene designated “AN80,”indicating that this gene is essential for survival. The AN80 amino acidand nucleic acid sequences are shown in FIG. 2 as SEQ ID NOs:3 and 4,respectively.

[0139] In a third strain, the mutation was in a gene designated “AN85,”indicating that this gene is essential for survival. The AN85 amino acidand nucleic acid sequences are shown in FIG. 3 as SEQ ID NOs:5 and 6,respectively.

[0140] In a fourth strain, the mutation was in a gene designated “AN17,”indicating that this gene is essential for survival. The AN17 amino acidand nucleic acid sequences are shown in FIG. 4 as SEQ ID NOs:7 and 8,respectively.

[0141] Now that each of these genes is known to be essential forsurvival of Aspergillus; the AN polypeptides (AN97, AN17, AN80, andAN85) can be used to identify antifungal agents by using the assaysdescribed herein. Other art-known assays to detect interactions of testcompounds with proteins, or to detect inhibition of fungal growth alsocan be used with the AN97, AN17, AN80, and AN85 genes and gene productsand homologs thereof.

Other Embodiments

[0142] The invention also features fragments, variants, analogs, andderivatives of the AN polypeptides described above that retain one ormore of the biological activities of the AN polypeptides, e.g., asdetermined in a complementation assay. Also included within theinvention are naturally-occurring and non-naturally-occurring allelicvariants. Compared with the naturally-occurring AN97, AN80, AN85, andAN17 nucleotide sequences depicted in FIGS. 1, 2, 3, and 4 respectively,the nucleic acid sequence encoding allelic variants may have asubstitution, deletion, or addition of one or more nucleotides. Thepreferred allelic variants are functionally equivalent to an ANpolypeptide, e.g., as determined in a complementation assay.

[0143] It is to be understood that, while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 35 <210> SEQ ID NO 1<211> LENGTH: 5596 <212> TYPE: DNA <213> ORGANISM: Aspergillus nidulans<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (604)...(2655) <221>NAME/KEY: CDS <222> LOCATION: (2706)...(3992) <400> SEQUENCE: 1agcgctgcgc agggcagctg tggcaaatcg ccggacgctt tggcgaaaca tcctgtcaat 60atcaatgctg ctcctgaaac agaaaaagac aagacgaagt tccccggatt gtatctcgaa 120tgaggggacc gatttccggc gttagtaaga ggtcacgtga aagatggcgt gctaactagt 180atgcaaggca tttcggctca ggcaaaatac ccagtcaaca atttgttgcc tggaggtgga 240aatacgagac ccttgattgc gagcagtgtg tgattaggat agctgaggca ttgtattcat 300gtatcaggaa cctgatcgtc aaagcgttgc aggctgctgg gctgggcacg tgctgcccta 360acccttatct atctactggt ttggggtgtt tgtttatgct ccgccccgtg actctcagca 420acggttataa cgagtagtgg cagcagccaa cgaacttctt tgctgccgac ctcacgccaa 480acaaaagcct ttactggaaa caggctgatc agcaaatcaa gatatactag gatgagttga 540tattatcacc ggccgcagat tactgacccg acacccttac tgcgtcatta cccctcgatc 600aag atg ccg agt cga gtt tcc gcc cgt tca aca tcc acc gcc tcg cgc 648 MetPro Ser Arg Val Ser Ala Arg Ser Thr Ser Thr Ala Ser Arg 1 5 10 15 aaaggc tct aca cag act gcg aca agc ggt cgc gct ggc tca gcg acc 696 Lys GlySer Thr Gln Thr Ala Thr Ser Gly Arg Ala Gly Ser Ala Thr 20 25 30 cca tcattc gcc atc cca gag gaa act gca tta ccc gag gct gtt cca 744 Pro Ser PheAla Ile Pro Glu Glu Thr Ala Leu Pro Glu Ala Val Pro 35 40 45 acc ctt cgccgc gat gta tgc gcc att ttc gcg gat gcc cag cgt tcg 792 Thr Leu Arg ArgAsp Val Cys Ala Ile Phe Ala Asp Ala Gln Arg Ser 50 55 60 act gcc ggt catcgc aaa ctt gtc gtc cga cta agg aaa atc cag gag 840 Thr Ala Gly His ArgLys Leu Val Val Arg Leu Arg Lys Ile Gln Glu 65 70 75 gtg tgc tgt gct ataccc cag aag aac tcc aaa aaa gac agt tca act 888 Val Cys Cys Ala Ile ProGln Lys Asn Ser Lys Lys Asp Ser Ser Thr 80 85 90 95 gaa gag cga ttg attccc ggc gaa gag acg gta cca gaa aag gag ttc 936 Glu Glu Arg Leu Ile ProGly Glu Glu Thr Val Pro Glu Lys Glu Phe 100 105 110 aac gtc gaa gta agtcgt tgt gtg ttg cgc atc ttg tct att aag aag 984 Asn Val Glu Val Ser ArgCys Val Leu Arg Ile Leu Ser Ile Lys Lys 115 120 125 aca gag cct gtt ggcgat cga atc ctg cgg ttt ctc ggg aac ttc ctt 1032 Thr Glu Pro Val Gly AspArg Ile Leu Arg Phe Leu Gly Asn Phe Leu 130 135 140 act cat gcc tcg gaaaag gac gct gag atc ttc ggc tct gaa gaa gat 1080 Thr His Ala Ser Glu LysAsp Ala Glu Ile Phe Gly Ser Glu Glu Asp 145 150 155 gaa gac gat atg cagaat tcg cac gaa aga ccg act gcc cac ttg acc 1128 Glu Asp Asp Met Gln AsnSer His Glu Arg Pro Thr Ala His Leu Thr 160 165 170 175 acc agt ctt gtctcc ctg tta gtg cct ttg ttg tct gca aaa gac aag 1176 Thr Ser Leu Val SerLeu Leu Val Pro Leu Leu Ser Ala Lys Asp Lys 180 185 190 gtt gtg cgc ttccgt acc acg caa att atc gcg cac atc gtc aat tca 1224 Val Val Arg Phe ArgThr Thr Gln Ile Ile Ala His Ile Val Asn Ser 195 200 205 ctc gat acc gtagac gac gaa tta tac cac act ctc cgg caa ggc ctt 1272 Leu Asp Thr Val AspAsp Glu Leu Tyr His Thr Leu Arg Gln Gly Leu 210 215 220 cta aaa cgg attcgc gac aaa gaa cct tcg gtg cgg gta caa gca gtg 1320 Leu Lys Arg Ile ArgAsp Lys Glu Pro Ser Val Arg Val Gln Ala Val 225 230 235 atg ggt ctc ggccgc ttg gcc gga aat gaa gag gac gat gac gaa aat 1368 Met Gly Leu Gly ArgLeu Ala Gly Asn Glu Glu Asp Asp Asp Glu Asn 240 245 250 255 gat gat accagt gcc ctt gtg gag aag ctc gtg gac ata atg caa aat 1416 Asp Asp Thr SerAla Leu Val Glu Lys Leu Val Asp Ile Met Gln Asn 260 265 270 gac acg gctgca gag gtt cgg agg aca tta ctc ctc aac ctc cca ttg 1464 Asp Thr Ala AlaGlu Val Arg Arg Thr Leu Leu Leu Asn Leu Pro Leu 275 280 285 att ccg tctacc ctt cca tac ctc ctc gaa cgc gcc cgt gac ctc gat 1512 Ile Pro Ser ThrLeu Pro Tyr Leu Leu Glu Arg Ala Arg Asp Leu Asp 290 295 300 gct ccc acacga agg gca tta tat tct cgt cta ctt ccg aca ctg gga 1560 Ala Pro Thr ArgArg Ala Leu Tyr Ser Arg Leu Leu Pro Thr Leu Gly 305 310 315 gat ttc cgacat tta tct ctc tcc atg aga gaa aag ttg ctc aga tgg 1608 Asp Phe Arg HisLeu Ser Leu Ser Met Arg Glu Lys Leu Leu Arg Trp 320 325 330 335 ggt cttcgt gat cgc gac aaa agt gtg agg aag gcc act gga aag ttg 1656 Gly Leu ArgAsp Arg Asp Lys Ser Val Arg Lys Ala Thr Gly Lys Leu 340 345 350 ttc tatgac cgc tgg att gag ata tcg ctg gca cga aca atg acc ctg 1704 Phe Tyr AspArg Trp Ile Glu Ile Ser Leu Ala Arg Thr Met Thr Leu 355 360 365 aga attcgg gca gcg ctc gga acg aga att ccc gct tta ctg gag ttg 1752 Arg Ile ArgAla Ala Leu Gly Thr Arg Ile Pro Ala Leu Leu Glu Leu 370 375 380 ttg gagcgt atc gat gtg gtg aac tca ggc atg gaa tcc ggc ata gcg 1800 Leu Glu ArgIle Asp Val Val Asn Ser Gly Met Glu Ser Gly Ile Ala 385 390 395 cac gaagct atg cgc agt ttc tgg gaa ggt cga cca gac tat cga gag 1848 His Glu AlaMet Arg Ser Phe Trp Glu Gly Arg Pro Asp Tyr Arg Glu 400 405 410 415 gcggta cta ttc gac gaa gcc ttc tgg gag tca atg aca gca gaa tcc 1896 Ala ValLeu Phe Asp Glu Ala Phe Trp Glu Ser Met Thr Ala Glu Ser 420 425 430 gctttc ctc ctt cgc tca ttc aat gac ttt tgc cgg gtt gaa aac gaa 1944 Ala PheLeu Leu Arg Ser Phe Asn Asp Phe Cys Arg Val Glu Asn Glu 435 440 445 ggtaaa tat gac agc ctc gcc gat gag aag atc cca gtc gtt aca gcc 1992 Gly LysTyr Asp Ser Leu Ala Asp Glu Lys Ile Pro Val Val Thr Ala 450 455 460 ctcgca atg tat ctt cat aag tac atg acc gag ctt ctg cag cgc aag 2040 Leu AlaMet Tyr Leu His Lys Tyr Met Thr Glu Leu Leu Gln Arg Lys 465 470 475 aagctc aca aag gat gct act gac gta aac gac gac gat acc gtc gaa 2088 Lys LeuThr Lys Asp Ala Thr Asp Val Asn Asp Asp Asp Thr Val Glu 480 485 490 495atc gaa ttt atc gtc gag caa ctg ctt cac atc gcg atg aca cta gac 2136 IleGlu Phe Ile Val Glu Gln Leu Leu His Ile Ala Met Thr Leu Asp 500 505 510tac agc gac gaa gtt ggg cgg cga aag atg ttt tct cta ctc cgt gag 2184 TyrSer Asp Glu Val Gly Arg Arg Lys Met Phe Ser Leu Leu Arg Glu 515 520 525gct ctc gct gtc cca gag ctc cct cag gaa tcg acc aag ctc gcg gtt 2232 AlaLeu Ala Val Pro Glu Leu Pro Gln Glu Ser Thr Lys Leu Ala Val 530 535 540gag aca ctg aga tgt gtt tgt ggg ccc gac gcc gcg gca gag agc gaa 2280 GluThr Leu Arg Cys Val Cys Gly Pro Asp Ala Ala Ala Glu Ser Glu 545 550 555ttc tgc agt gtt gtt ctg gaa gcc att gct gaa gtt cat gac aca atc 2328 PheCys Ser Val Val Leu Glu Ala Ile Ala Glu Val His Asp Thr Ile 560 565 570575 agc acc gag gat agt ttc gtt tct gca aag tct gag att agc gat gat 2376Ser Thr Glu Asp Ser Phe Val Ser Ala Lys Ser Glu Ile Ser Asp Asp 580 585590 gcc agc agc cgc caa cga tcc gaa acg ccg atg agt gaa gat gac aag 2424Ala Ser Ser Arg Gln Arg Ser Glu Thr Pro Met Ser Glu Asp Asp Lys 595 600605 cca ttc aac aag gag gag gca aag gct aag gtc ctc aag gaa atc gtt 2472Pro Phe Asn Lys Glu Glu Ala Lys Ala Lys Val Leu Lys Glu Ile Val 610 615620 att aat atg aag tgt ctg cac att gcc ctt tgc atg ctc cag aat gtt 2520Ile Asn Met Lys Cys Leu His Ile Ala Leu Cys Met Leu Gln Asn Val 625 630635 gaa ggc aac ctg caa gca aat atg aat ctg gtg acc atg ttg aat aac 2568Glu Gly Asn Leu Gln Ala Asn Met Asn Leu Val Thr Met Leu Asn Asn 640 645650 655 ttg gta gta cct gct gtt cgg agc cac gaa gcg cca att cga gag cgc2616 Leu Val Val Pro Ala Val Arg Ser His Glu Ala Pro Ile Arg Glu Arg 660665 670 ggt ctc gaa tgt ctt ggg ctg tgc tgc ttg ctg gac aag gtaagttcca2665 Gly Leu Glu Cys Leu Gly Leu Cys Cys Leu Leu Asp Lys 675 680tccttactaa atacatcttc ttctctaacc tctctgttag act ctc gca gaa gaa 2720 ThrLeu Ala Glu Glu 685 aat atg acg ctg ttt att cac tgt tac agc aag ggc cacgaa aac cta 2768 Asn Met Thr Leu Phe Ile His Cys Tyr Ser Lys Gly His GluAsn Leu 690 695 700 705 cag gtc act gct att cat atc ctt tgc gat atg ttaatt agc cat cct 2816 Gln Val Thr Ala Ile His Ile Leu Cys Asp Met Leu IleSer His Pro 710 715 720 tcg ctg gtg gct ccc gtt acc cag gcc gat aag gagaca gtt gcg cca 2864 Ser Leu Val Ala Pro Val Thr Gln Ala Asp Lys Glu ThrVal Ala Pro 725 730 735 ccg gcg ttc cag aag cca ctg ctt aag gtc ttt tccaga gct ctc aaa 2912 Pro Ala Phe Gln Lys Pro Leu Leu Lys Val Phe Ser ArgAla Leu Lys 740 745 750 cca aat tca ccc gcg tct gta caa acg gca gct gcgaca gct ctt tct 2960 Pro Asn Ser Pro Ala Ser Val Gln Thr Ala Ala Ala ThrAla Leu Ser 755 760 765 aag ctt ctg ctc act ggt gtt ttt act cca tct gccgcc aat atc ccc 3008 Lys Leu Leu Leu Thr Gly Val Phe Thr Pro Ser Ala AlaAsn Ile Pro 770 775 780 785 gat gcc att caa gag ttc aac caa cat gcc atcgaa aca ctg cta cag 3056 Asp Ala Ile Gln Glu Phe Asn Gln His Ala Ile GluThr Leu Leu Gln 790 795 800 tcc ctc gtt gtc tcc ttc ttc cat ccc cga actcgc gag aat ccc gca 3104 Ser Leu Val Val Ser Phe Phe His Pro Arg Thr ArgGlu Asn Pro Ala 805 810 815 ctc cga cag gca ctc gcg tac ttc ttc cct gtctac tgc cac tcc cgg 3152 Leu Arg Gln Ala Leu Ala Tyr Phe Phe Pro Val TyrCys His Ser Arg 820 825 830 ccg gat aac acc cag cat atg aga aag att actgta cct gtc atc cgg 3200 Pro Asp Asn Thr Gln His Met Arg Lys Ile Thr ValPro Val Ile Arg 835 840 845 acc atc cta aac tca gcg gaa gaa tac tac tcactt gag gct gaa gag 3248 Thr Ile Leu Asn Ser Ala Glu Glu Tyr Tyr Ser LeuGlu Ala Glu Glu 850 855 860 865 gac agt gat ggt gat att gat gag tct gttggg gag aag gaa ttg aag 3296 Asp Ser Asp Gly Asp Ile Asp Glu Ser Val GlyGlu Lys Glu Leu Lys 870 875 880 gcc ctg atg agc gga gtt ctt ggt atg cttgcg gag tgg acg gat gag 3344 Ala Leu Met Ser Gly Val Leu Gly Met Leu AlaGlu Trp Thr Asp Glu 885 890 895 cga aga gtg atc gga ctt ggc ggc gaa cgggtc ctt gct ggg ggc ctt 3392 Arg Arg Val Ile Gly Leu Gly Gly Glu Arg ValLeu Ala Gly Gly Leu 900 905 910 gct agc tcc aat gtt tgt ggc att atc cacttg caa ctg att aag gac 3440 Ala Ser Ser Asn Val Cys Gly Ile Ile His LeuGln Leu Ile Lys Asp 915 920 925 ata ctg gaa cga gtg ctc ggg atc agt gaaggc agc aat cgc tgc tct 3488 Ile Leu Glu Arg Val Leu Gly Ile Ser Glu GlySer Asn Arg Cys Ser 930 935 940 945 aaa caa caa cga aaa ctc ctg ttt tcactc atg agc aag ctc tat att 3536 Lys Gln Gln Arg Lys Leu Leu Phe Ser LeuMet Ser Lys Leu Tyr Ile 950 955 960 gcg ccg cca acg gca ctt tcg cgc tcagcg tcc cag gcc ccc gaa gac 3584 Ala Pro Pro Thr Ala Leu Ser Arg Ser AlaSer Gln Ala Pro Glu Asp 965 970 975 gac tcg ttc cgt tcc agc gtg cga agctcc cat ggc gaa ctc aat ccc 3632 Asp Ser Phe Arg Ser Ser Val Arg Ser SerHis Gly Glu Leu Asn Pro 980 985 990 gaa aac ctt gcc ctc gcg cag gaa gtcaag gag cta ctt gac cag acc 3680 Glu Asn Leu Ala Leu Ala Gln Glu Val LysGlu Leu Leu Asp Gln Thr 995 1000 1005 atc gaa gaa ggt gtg gcg gct gatgct gct agc cga aat gcc ctc gtc 3728 Ile Glu Glu Gly Val Ala Ala Asp AlaAla Ser Arg Asn Ala Leu Val 1010 1015 1020 1025 aag gtg aag aac gtg gtgctc aag cta ctg gcg gct ccc atg cga cct 3776 Lys Val Lys Asn Val Val LeuLys Leu Leu Ala Ala Pro Met Arg Pro 1030 1035 1040 tct agc gca cgc ggccgc gag agc agt gtc gaa agt gac att ggc agt 3824 Ser Ser Ala Arg Gly ArgGlu Ser Ser Val Glu Ser Asp Ile Gly Ser 1045 1050 1055 gtt cga tct tccaga agt gtt cgg ccg tcc gta gag cct ggc ttt ggg 3872 Val Arg Ser Ser ArgSer Val Arg Pro Ser Val Glu Pro Gly Phe Gly 1060 1065 1070 cgc cgc ggtgta tcc gtg gag ccc agt atc atg gag gag gat gag aat 3920 Arg Arg Gly ValSer Val Glu Pro Ser Ile Met Glu Glu Asp Glu Asn 1075 1080 1085 gag gatagc cgg gcg act ctg gac agt aga atg act gtt atc aaa gag 3968 Glu Asp SerArg Ala Thr Leu Asp Ser Arg Met Thr Val Ile Lys Glu 1090 1095 1100 1105gag gat gcc gac gct atg gag gaa tgattttcgg tctcaagatc tttgctgtct 4022Glu Asp Ala Asp Ala Met Glu Glu 1110 ggttcggcgt tggggaggtt tcccggcagggctaatggtc atatttatgg ttaggttgcg 4082 atgtaattat tcgattcttg gttatgcttgaacatgctct atatgttaca aataattcac 4142 tccaaacgtt catgtatgag tatggatctgttttatattg gccttaccag gatagctcag 4202 ttcttggcga agttatccca gactgacagctgcctccagg ccagaattgg ctagtcttag 4262 tcttaggtag catctgagtt atcgcgtggtatcaacagtg atcagtgtgg aagggccatc 4322 cgatctgttt gatcttacca gaacgtgttacaacaattca acccaccata tatatggtat 4382 ctacgtcaat gtgaatgaat ctgcttgggcagccttatga ctctggtgac gcgactcggg 4442 gcttgattca atgcgggcaa gaccgcatgtggagactcct agcatcggat gtgaggcttc 4502 cgttttaatt tcttcctcca aatcgtctgcctgcctcgct gctttgaaat actccggagg 4562 taccaaagta aagataaatg gttgactctgagagactgct ttgacctcct ggaccaagtc 4622 gtgcctagcc agaaggggag tgttcaatgggctttgtgag gctactaagg ccgcacgata 4682 caccggagat gcaaagaagt ccgatacggtcgtccatatc tcgagcacct ttattactgg 4742 cgcttttgca gttatatgga ggcgtttaatgattgcgtgt tcggaatccg atgaataata 4802 tctcattagt cgactaaacg gggatgaggatggatgactg ctggtatctt ggtctcaaac 4862 tgtaataagc gtctcggcaa caccgtacggttgacaatcc tgggcagatg gcagcacctg 4922 tagaatccaa gaagacgcag ctggactcattgagacagtt gaattcctta actataatga 4982 cagactaata atacaaaagt gcggtggtcaacttcttccc aatcccctca aaagtcagac 5042 ccgaccctgt tctttctaat aatctgacgctccaccaaaa gtccagcttc tgggcgactt 5102 tctttttctt ccccatcctt ttcctttcccactctcctcc ctcctctctc gcttctcttc 5162 ctttcgctgt atgttttttg ttgcttgattcacgactttc tttttccttc tggtcgtgga 5222 tccgtgtctt ctgcccccac ttgcagaggcacgatttttc tccctctccc tctcctccct 5282 tccgtactcc ccccctcccc cctgctctgcgcctttggca tccggagcct gcgtcgagac 5342 cgtgagcgat ggcctccgtg tcagctcccacgcccaagct ggaccgctac atcgtcgttc 5402 atgtggcaac tacctgcgat gagcatggcgtctacgtcac caaggactct gcagagtgat 5462 cgagttgggg tggatcttgt tggataccaaaacctgcgag agtcgcagtg attctctccc 5522 tgcaccacac ctattccacc ccctcttttgtgtcttgatt ctcgccggcc taccgggatt 5582 ctgccgacga catt 5596 <210> SEQ IDNO 2 <211> LENGTH: 684 <212> TYPE: PRT <213> ORGANISM: Aspergillusnidulans <400> SEQUENCE: 2 Met Pro Ser Arg Val Ser Ala Arg Ser Thr SerThr Ala Ser Arg Lys 1 5 10 15 Gly Ser Thr Gln Thr Ala Thr Ser Gly ArgAla Gly Ser Ala Thr Pro 20 25 30 Ser Phe Ala Ile Pro Glu Glu Thr Ala LeuPro Glu Ala Val Pro Thr 35 40 45 Leu Arg Arg Asp Val Cys Ala Ile Phe AlaAsp Ala Gln Arg Ser Thr 50 55 60 Ala Gly His Arg Lys Leu Val Val Arg LeuArg Lys Ile Gln Glu Val 65 70 75 80 Cys Cys Ala Ile Pro Gln Lys Asn SerLys Lys Asp Ser Ser Thr Glu 85 90 95 Glu Arg Leu Ile Pro Gly Glu Glu ThrVal Pro Glu Lys Glu Phe Asn 100 105 110 Val Glu Val Ser Arg Cys Val LeuArg Ile Leu Ser Ile Lys Lys Thr 115 120 125 Glu Pro Val Gly Asp Arg IleLeu Arg Phe Leu Gly Asn Phe Leu Thr 130 135 140 His Ala Ser Glu Lys AspAla Glu Ile Phe Gly Ser Glu Glu Asp Glu 145 150 155 160 Asp Asp Met GlnAsn Ser His Glu Arg Pro Thr Ala His Leu Thr Thr 165 170 175 Ser Leu ValSer Leu Leu Val Pro Leu Leu Ser Ala Lys Asp Lys Val 180 185 190 Val ArgPhe Arg Thr Thr Gln Ile Ile Ala His Ile Val Asn Ser Leu 195 200 205 AspThr Val Asp Asp Glu Leu Tyr His Thr Leu Arg Gln Gly Leu Leu 210 215 220Lys Arg Ile Arg Asp Lys Glu Pro Ser Val Arg Val Gln Ala Val Met 225 230235 240 Gly Leu Gly Arg Leu Ala Gly Asn Glu Glu Asp Asp Asp Glu Asn Asp245 250 255 Asp Thr Ser Ala Leu Val Glu Lys Leu Val Asp Ile Met Gln AsnAsp 260 265 270 Thr Ala Ala Glu Val Arg Arg Thr Leu Leu Leu Asn Leu ProLeu Ile 275 280 285 Pro Ser Thr Leu Pro Tyr Leu Leu Glu Arg Ala Arg AspLeu Asp Ala 290 295 300 Pro Thr Arg Arg Ala Leu Tyr Ser Arg Leu Leu ProThr Leu Gly Asp 305 310 315 320 Phe Arg His Leu Ser Leu Ser Met Arg GluLys Leu Leu Arg Trp Gly 325 330 335 Leu Arg Asp Arg Asp Lys Ser Val ArgLys Ala Thr Gly Lys Leu Phe 340 345 350 Tyr Asp Arg Trp Ile Glu Ile SerLeu Ala Arg Thr Met Thr Leu Arg 355 360 365 Ile Arg Ala Ala Leu Gly ThrArg Ile Pro Ala Leu Leu Glu Leu Leu 370 375 380 Glu Arg Ile Asp Val ValAsn Ser Gly Met Glu Ser Gly Ile Ala His 385 390 395 400 Glu Ala Met ArgSer Phe Trp Glu Gly Arg Pro Asp Tyr Arg Glu Ala 405 410 415 Val Leu PheAsp Glu Ala Phe Trp Glu Ser Met Thr Ala Glu Ser Ala 420 425 430 Phe LeuLeu Arg Ser Phe Asn Asp Phe Cys Arg Val Glu Asn Glu Gly 435 440 445 LysTyr Asp Ser Leu Ala Asp Glu Lys Ile Pro Val Val Thr Ala Leu 450 455 460Ala Met Tyr Leu His Lys Tyr Met Thr Glu Leu Leu Gln Arg Lys Lys 465 470475 480 Leu Thr Lys Asp Ala Thr Asp Val Asn Asp Asp Asp Thr Val Glu Ile485 490 495 Glu Phe Ile Val Glu Gln Leu Leu His Ile Ala Met Thr Leu AspTyr 500 505 510 Ser Asp Glu Val Gly Arg Arg Lys Met Phe Ser Leu Leu ArgGlu Ala 515 520 525 Leu Ala Val Pro Glu Leu Pro Gln Glu Ser Thr Lys LeuAla Val Glu 530 535 540 Thr Leu Arg Cys Val Cys Gly Pro Asp Ala Ala AlaGlu Ser Glu Phe 545 550 555 560 Cys Ser Val Val Leu Glu Ala Ile Ala GluVal His Asp Thr Ile Ser 565 570 575 Thr Glu Asp Ser Phe Val Ser Ala LysSer Glu Ile Ser Asp Asp Ala 580 585 590 Ser Ser Arg Gln Arg Ser Glu ThrPro Met Ser Glu Asp Asp Lys Pro 595 600 605 Phe Asn Lys Glu Glu Ala LysAla Lys Val Leu Lys Glu Ile Val Ile 610 615 620 Asn Met Lys Cys Leu HisIle Ala Leu Cys Met Leu Gln Asn Val Glu 625 630 635 640 Gly Asn Leu GlnAla Asn Met Asn Leu Val Thr Met Leu Asn Asn Leu 645 650 655 Val Val ProAla Val Arg Ser His Glu Ala Pro Ile Arg Glu Arg Gly 660 665 670 Leu GluCys Leu Gly Leu Cys Cys Leu Leu Asp Lys 675 680 <210> SEQ ID NO 3 <211>LENGTH: 5596 <212> TYPE: DNA <213> ORGANISM: Aspergillus nidulans <400>SEQUENCE: 3 aatgtcgtcg gcagaatccc ggtaggccgg cgagaatcaa gacacaaaagagggggtgga 60 ataggtgtgg tgcagggaga gaatcactgc gactctcgca ggttttggtatccaacaaga 120 tccaccccaa ctcgatcact ctgcagagtc cttggtgacg tagacgccatgctcatcgca 180 ggtagttgcc acatgaacga cgatgtagcg gtccagcttg ggcgtgggagctgacacgga 240 ggccatcgct cacggtctcg acgcaggctc cggatgccaa aggcgcagagcaggggggag 300 ggggggagta cggaagggag gagagggaga gggagaaaaa tcgtgcctctgcaagtgggg 360 gcagaagaca cggatccacg accagaagga aaaagaaagt cgtgaatcaagcaacaaaaa 420 acatacagcg aaaggaagag aagcgagaga ggagggagga gagtgggaaaggaaaaggat 480 ggggaagaaa aagaaagtcg cccagaagct ggacttttgg tggagcgtcagattattaga 540 aagaacaggg tcgggtctga cttttgaggg gattgggaag aagttgaccaccgcactttt 600 gtattattag tctgtcatta tagttaagga attcaactgt ctcaatgagtccagctgcgt 660 cttcttggat tctacaggtg ctgccatctg cccaggattg tcaaccgtacggtgttgccg 720 agacgcttat tacagtttga gaccaagata ccagcagtca tccatcctcatccccgttta 780 gtcgactaat gagatattat tcatcggatt ccgaacacgc aatcattaaacgcctccata 840 taactgcaaa agcgccagta ataaaggtgc tcgagatatg gacgaccgtatcggacttct 900 ttgcatctcc ggtgtatcgt gcggccttag tagcctcaca aagcccattgaacactcccc 960 ttctggctag gcacgacttg gtccaggagg tcaaagcagt ctctcagagtcaaccattta 1020 tctttacttt ggtacctccg gagtatttca aagcagcgag gcaggcagacgatttggagg 1080 aagaaattaa aacggaagcc tcacatccga tgctaggagt ctccacatgcggtcttgccc 1140 gcattgaatc aagccccgag tcgcgtcacc agagtcataa ggctgcccaagcagattcat 1200 tcacattgac gtagatacca tatatatggt gggttgaatt gttgtaacacgttctggtaa 1260 gatcaaacag atcggatggc ccttccacac tgatcactgt tgataccacgcgataactca 1320 gatgctacct aagactaaga ctagccaatt ctggcctgga ggcagctgtcagtctgggat 1380 aacttcgcca agaactgagc tatcctggta aggccaatat aaaacagatccatactcata 1440 catgaacgtt tggagtgaat tatttgtaac atatagagca tgttcaagcataaccaagaa 1500 tcgaataatt acatcgcaac ctaaccataa atatgaccat tagccctgccgggaaacctc 1560 cccaacgccg aaccagacag caaagatctt gagaccgaaa atcattcctccatagcgtcg 1620 gcatcctcct ctttgataac agtcattcta ctgtccagag tcgcccggctatcctcattc 1680 tcatcctcct ccatgatact gggctccacg gatacaccgc ggcgcccaaagccaggctct 1740 acggacggcc gaacacttct ggaagatcga acactgccaa tgtcactttcgacactgctc 1800 tcgcggccgc gtgcgctaga aggtcgcatg ggagccgcca gtagcttgagcaccacgttc 1860 ttcaccttga cgagggcatt tcggctagca gcatcagccg ccacaccttcttcgatggtc 1920 tggtcaagta gctccttgac ttcctgcgcg agggcaaggt tttcgggattgagttcgcca 1980 tgggagcttc gcacgctgga acggaacgag tcgtcttcgg gggcctgggacgctgagcgc 2040 gaaagtgccg ttggcggcgc aatatagagc ttgctcatga gtgaaaacaggagttttcgt 2100 tgttgtttag agcagcgatt gctgccttca ctgatcccga gcactcgttccagtatgtcc 2160 ttaatcagtt gcaagtggat aatgccacaa acattggagc tagcaaggcccccagcaagg 2220 acccgttcgc cgccaagtcc gatcactctt cgctcatccg tccactccgcaagcatacca 2280 agaactccgc tcatcagggc cttcaattcc ttctccccaa cagactcatcaatatcacca 2340 tcactgtcct cttcagcctc aagtgagtag tattcttccg ctgagtttaggatggtccgg 2400 atgacaggta cagtaatctt tctcatatgc tgggtgttat ccggccgggagtggcagtag 2460 acagggaaga agtacgcgag tgcctgtcgg agtgcgggat tctcgcgagttcggggatgg 2520 aagaaggaga caacgaggga ctgtagcagt gtttcgatgg catgttggttgaactcttga 2580 atggcatcgg ggatattggc ggcagatgga gtaaaaacac cagtgagcagaagcttagaa 2640 agagctgtcg cagctgccgt ttgtacagac gcgggtgaat ttggtttgagagctctggaa 2700 aagaccttaa gcagtggctt ctggaacgcc ggtggcgcaa ctgtctccttatcggcctgg 2760 gtaacgggag ccaccagcga aggatggcta attaacatat cgcaaaggatatgaatagca 2820 gtgacctgta ggttttcgtg gcccttgctg taacagtgaa taaacagcgtcatattttct 2880 tctgcgagag tctaacagag aggttagaga agaagatgta tttagtaaggatggaactta 2940 ccttgtccag caagcagcac agcccaagac attcgagacc gcgctctcgaattggcgctt 3000 cgtggctccg aacagcaggt actaccaagt tattcaacat ggtcaccagattcatatttg 3060 cttgcaggtt gccttcaaca ttctggagca tgcaaagggc aatgtgcagacacttcatat 3120 taataacgat ttccttgagg accttagcct ttgcctcctc cttgttgaatggcttgtcat 3180 cttcactcat cggcgtttcg gatcgttggc ggctgctggc atcatcgctaatctcagact 3240 ttgcagaaac gaaactatcc tcggtgctga ttgtgtcatg aacttcagcaatggcttcca 3300 gaacaacact gcagaattcg ctctctgccg cggcgtcggg cccacaaacacatctcagtg 3360 tctcaaccgc gagcttggtc gattcctgag ggagctctgg gacagcgagagcctcacgga 3420 gtagagaaaa catctttcgc cgcccaactt cgtcgctgta gtctagtgtcatcgcgatgt 3480 gaagcagttg ctcgacgata aattcgattt cgacggtatc gtcgtcgtttacgtcagtag 3540 catcctttgt gagcttcttg cgctgcagaa gctcggtcat gtacttatgaagatacattg 3600 cgagggctgt aacgactggg atcttctcat cggcgaggct gtcatatttaccttcgtttt 3660 caacccggca aaagtcattg aatgagcgaa ggaggaaagc ggattctgctgtcattgact 3720 cccagaaggc ttcgtcgaat agtaccgcct ctcgatagtc tggtcgaccttcccagaaac 3780 tgcgcatagc ttcgtgcgct atgccggatt ccatgcctga gttcaccacatcgatacgct 3840 ccaacaactc cagtaaagcg ggaattctcg ttccgagcgc tgcccgaattctcagggtca 3900 ttgttcgtgc cagcgatatc tcaatccagc ggtcatagaa caactttccagtggccttcc 3960 tcacactttt gtcgcgatca cgaagacccc atctgagcaa cttttctctcatggagagag 4020 ataaatgtcg gaaatctccc agtgtcggaa gtagacgaga atataatgcccttcgtgtgg 4080 gagcatcgag gtcacgggcg cgttcgagga ggtatggaag ggtagacggaatcaatggga 4140 ggttgaggag taatgtcctc cgaacctctg cagccgtgtc attttgcattatgtccacga 4200 gcttctccac aagggcactg gtatcatcat tttcgtcatc gtcctcttcatttccggcca 4260 agcggccgag acccatcact gcttgtaccc gcaccgaagg ttctttgtcgcgaatccgtt 4320 ttagaaggcc ttgccggaga gtgtggtata attcgtcgtc tacggtatcgagtgaattga 4380 cgatgtgcgc gataatttgc gtggtacgga agcgcacaac cttgtcttttgcagacaaca 4440 aaggcactaa cagggagaca agactggtgg tcaagtgggc agtcggtctttcgtgcgaat 4500 tctgcatatc gtcttcatct tcttcagagc cgaagatctc agcgtccttttccgaggcat 4560 gagtaaggaa gttcccgaga aaccgcagga ttcgatcgcc aacaggctctgtcttcttaa 4620 tagacaagat gcgcaacaca caacgactta cttcgacgtt gaactccttttctggtaccg 4680 tctcttcgcc gggaatcaat cgctcttcag ttgaactgtc ttttttggagttcttctggg 4740 gtatagcaca gcacacctcc tggattttcc ttagtcggac gacaagtttgcgatgaccgg 4800 cagtcgaacg ctgggcatcc gcgaaaatgg cgcatacatc gcggcgaagggttggaacag 4860 cctcgggtaa tgcagtttcc tctgggatgg cgaatgatgg ggtcgctgagccagcgcgac 4920 cgcttgtcgc agtctgtgta gagcctttgc gcgaggcggt ggatgttgaacgggcggaaa 4980 ctcgactcgg catcttgatc gaggggtaat gacgcagtaa gggtgtcgggtcagtaatct 5040 gcggccggtg ataatatcaa ctcatcctag tatatcttga tttgctgatcagcctgtttc 5100 cagtaaaggc ttttgtttgg cgtgaggtcg gcagcaaaga agttcgttggctgctgccac 5160 tactcgttat aaccgttgct gagagtcacg gggcggagca taaacaaacaccccaaacca 5220 gtagatagat aagggttagg gcagcacgtg cccagcccag cagcctgcaacgctttgacg 5280 atcaggttcc tgatacatga atacaatgcc tcagctatcc taatcacacactgctcgcaa 5340 tcaagggtct cgtatttcca cctccaggca acaaattgtt gactgggtattttgcctgag 5400 ccgaaatgcc ttgcatacta gttagcacgc catctttcac gtgacctcttactaacgccg 5460 gaaatcggtc ccctcattcg agatacaatc cggggaactt cgtcttgtctttttctgttt 5520 caggagcagc attgatattg acaggatgtt tcgccaaagc gtccggcgatttgccacagc 5580 tgccctgcgc agcgct 5596 <210> SEQ ID NO 4 <211> LENGTH:1758 <212> TYPE: DNA <213> ORGANISM: Aspergillus nidulans <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (162)...(1319) <400> SEQUENCE: 4caaaagtctt gatcacaggg gcacaagcgc aattgagcca ccatgcttac ggacggcatc 60gaaggggtca aggagaaagt ctttgtgctc gtgaccggtg ccaacaggta cagtgaaacc 120ctgcgctctg tctcctatct catgcggtcc gttagtggtt t atg ttt cta act gtt 176Met Phe Leu Thr Val 1 5 acc cct tgt ggg ttt tca ccg ttt agc gga cta ggatac tca acg tgt 224 Thr Pro Cys Gly Phe Ser Pro Phe Ser Gly Leu Gly TyrSer Thr Cys 10 15 20 tgc cgt ctt gca gat gaa ttc ctg gcg tct cat cgg aacgac cat cgt 272 Cys Arg Leu Ala Asp Glu Phe Leu Ala Ser His Arg Asn AspHis Arg 25 30 35 tca ttg aca atc atc ttc act acc cgg agc aca aga aag ggaagc gac 320 Ser Leu Thr Ile Ile Phe Thr Thr Arg Ser Thr Arg Lys Gly SerAsp 40 45 50 acc ctt cgc aac cta cag aat cac ctc cgc acc tcc acc ttc ggtgct 368 Thr Leu Arg Asn Leu Gln Asn His Leu Arg Thr Ser Thr Phe Gly Ala55 60 65 tcg gcc acc gct cga gtg acc ttc gtt cct gaa aat gtc gac ctc tgc416 Ser Ala Thr Ala Arg Val Thr Phe Val Pro Glu Asn Val Asp Leu Cys 7075 80 85 aac ctc ctc tcg gtc cgc gcg cta tcc cgt cgc ctg aac aag acc ttc464 Asn Leu Leu Ser Val Arg Ala Leu Ser Arg Arg Leu Asn Lys Thr Phe 9095 100 cca aaa ctc gac gcg att gtg ctt aat gcc ggg ata ggg ggt tgg tct512 Pro Lys Leu Asp Ala Ile Val Leu Asn Ala Gly Ile Gly Gly Trp Ser 105110 115 ggc ctc aat tgg cct ctg gcc gta tgg agc gtt tgc acc gac att atc560 Gly Leu Asn Trp Pro Leu Ala Val Trp Ser Val Cys Thr Asp Ile Ile 120125 130 cat gcg acg acg tgg cca aag tac aaa att gcg cct gta ggt ctc ata608 His Ala Thr Thr Trp Pro Lys Tyr Lys Ile Ala Pro Val Gly Leu Ile 135140 145 acg gac aac cag aca att act gtg acc gac aag gag ccc cgc ctg gga656 Thr Asp Asn Gln Thr Ile Thr Val Thr Asp Lys Glu Pro Arg Leu Gly 150155 160 165 acc gtc ttc tgc gcc aac gtc ttc ggc cac tac atg ctc gcg cataat 704 Thr Val Phe Cys Ala Asn Val Phe Gly His Tyr Met Leu Ala His Asn170 175 180 gtc atg cct ctc ctg cac cga tcc gga tcc ccc aac gga ccc ggacgc 752 Val Met Pro Leu Leu His Arg Ser Gly Ser Pro Asn Gly Pro Gly Arg185 190 195 gtg ata tgg ctc tcc agc act gaa gcc acg atc aac ttc ttc gatgtt 800 Val Ile Trp Leu Ser Ser Thr Glu Ala Thr Ile Asn Phe Phe Asp Val200 205 210 gat gat ttt cag gcg ctc cgg tcc aaa gct ccc tac gag tca tcaaaa 848 Asp Asp Phe Gln Ala Leu Arg Ser Lys Ala Pro Tyr Glu Ser Ser Lys215 220 225 gcg cta aca gac ctc cta tcc ctc acc tca gac ctt ccc agt actgct 896 Ala Leu Thr Asp Leu Leu Ser Leu Thr Ser Asp Leu Pro Ser Thr Ala230 235 240 245 ccc tgg gtg aaa agc ttc tat tcc acc gac ttc gaa acc gattcc aag 944 Pro Trp Val Lys Ser Phe Tyr Ser Thr Asp Phe Glu Thr Asp SerLys 250 255 260 ccc agc acc gga cct gag acc gcc tcg acc ata ccc aac gtatac ctc 992 Pro Ser Thr Gly Pro Glu Thr Ala Ser Thr Ile Pro Asn Val TyrLeu 265 270 275 tct cac ccc gga atc tgc gct acg gcg att ata ccc ctt cctaca atc 1040 Ser His Pro Gly Ile Cys Ala Thr Ala Ile Ile Pro Leu Pro ThrIle 280 285 290 ctc atc tac gca atg gtc gcc gca ttt tgg cta gcc cgc atcctc ggc 1088 Leu Ile Tyr Ala Met Val Ala Ala Phe Trp Leu Ala Arg Ile LeuGly 295 300 305 tcc cct tgg cat acc tta tcc acc tac cta ggc gct tgc agccct gtc 1136 Ser Pro Trp His Thr Leu Ser Thr Tyr Leu Gly Ala Cys Ser ProVal 310 315 320 325 tgg ctt gct ctc tcc aca caa tca gaa ctc gac gcc gccgaa gca ccg 1184 Trp Leu Ala Leu Ser Thr Gln Ser Glu Leu Asp Ala Ala GluAla Pro 330 335 340 tac cgg aaa cac ggc ggc ggc agg gtg aaa tgg ggg tcttcg gcg tct 1232 Tyr Arg Lys His Gly Gly Gly Arg Val Lys Trp Gly Ser SerAla Ser 345 350 355 cga tta ggt gta gcc tcc gtc gta tct tcg gag gtt gacgga tgg ggc 1280 Arg Leu Gly Val Ala Ser Val Val Ser Ser Glu Val Asp GlyTrp Gly 360 365 370 tat ggg ggt gtt cct ggg gcc ggc tgt tgt ggc gga ggatagggtctga 1329 Tyr Gly Gly Val Pro Gly Ala Gly Cys Cys Gly Gly Gly 375380 385 aggcgcaagc gtggtgcagt ggatcttacg gctgagggga aggagggattccaggaactg 1389 ggggctatat gttggaggca gatggaggag ctgaggatcc tgtgggataacttacttgat 1449 gaagagagaa ggggactggt gtgacggcgt aggtggcttg tcctgggagtgagatctctt 1509 acatttcggc cttcgtccct aaaatccttt tctcccttcc tctttattatacgatgtcgg 1569 cggttttatg ttcaatacag cacatctacg gtacaaagac aacatatagctaatataata 1629 tcatagataa tagtaataat caagcacaaa agctcgattc tgcaagatctcaatatcttt 1689 attccagttt tcactgctct tgtcttccat atttacattc cacgtccacgtgcatccttt 1749 aaaaacagt 1758 <210> SEQ ID NO 5 <211> LENGTH: 386 <212>TYPE: PRT <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 5 Met PheLeu Thr Val Thr Pro Cys Gly Phe Ser Pro Phe Ser Gly Leu 1 5 10 15 GlyTyr Ser Thr Cys Cys Arg Leu Ala Asp Glu Phe Leu Ala Ser His 20 25 30 ArgAsn Asp His Arg Ser Leu Thr Ile Ile Phe Thr Thr Arg Ser Thr 35 40 45 ArgLys Gly Ser Asp Thr Leu Arg Asn Leu Gln Asn His Leu Arg Thr 50 55 60 SerThr Phe Gly Ala Ser Ala Thr Ala Arg Val Thr Phe Val Pro Glu 65 70 75 80Asn Val Asp Leu Cys Asn Leu Leu Ser Val Arg Ala Leu Ser Arg Arg 85 90 95Leu Asn Lys Thr Phe Pro Lys Leu Asp Ala Ile Val Leu Asn Ala Gly 100 105110 Ile Gly Gly Trp Ser Gly Leu Asn Trp Pro Leu Ala Val Trp Ser Val 115120 125 Cys Thr Asp Ile Ile His Ala Thr Thr Trp Pro Lys Tyr Lys Ile Ala130 135 140 Pro Val Gly Leu Ile Thr Asp Asn Gln Thr Ile Thr Val Thr AspLys 145 150 155 160 Glu Pro Arg Leu Gly Thr Val Phe Cys Ala Asn Val PheGly His Tyr 165 170 175 Met Leu Ala His Asn Val Met Pro Leu Leu His ArgSer Gly Ser Pro 180 185 190 Asn Gly Pro Gly Arg Val Ile Trp Leu Ser SerThr Glu Ala Thr Ile 195 200 205 Asn Phe Phe Asp Val Asp Asp Phe Gln AlaLeu Arg Ser Lys Ala Pro 210 215 220 Tyr Glu Ser Ser Lys Ala Leu Thr AspLeu Leu Ser Leu Thr Ser Asp 225 230 235 240 Leu Pro Ser Thr Ala Pro TrpVal Lys Ser Phe Tyr Ser Thr Asp Phe 245 250 255 Glu Thr Asp Ser Lys ProSer Thr Gly Pro Glu Thr Ala Ser Thr Ile 260 265 270 Pro Asn Val Tyr LeuSer His Pro Gly Ile Cys Ala Thr Ala Ile Ile 275 280 285 Pro Leu Pro ThrIle Leu Ile Tyr Ala Met Val Ala Ala Phe Trp Leu 290 295 300 Ala Arg IleLeu Gly Ser Pro Trp His Thr Leu Ser Thr Tyr Leu Gly 305 310 315 320 AlaCys Ser Pro Val Trp Leu Ala Leu Ser Thr Gln Ser Glu Leu Asp 325 330 335Ala Ala Glu Ala Pro Tyr Arg Lys His Gly Gly Gly Arg Val Lys Trp 340 345350 Gly Ser Ser Ala Ser Arg Leu Gly Val Ala Ser Val Val Ser Ser Glu 355360 365 Val Asp Gly Trp Gly Tyr Gly Gly Val Pro Gly Ala Gly Cys Cys Gly370 375 380 ly Gly 385 <210> SEQ ID NO 6 <211> LENGTH: 1758 <212> TYPE:DNA <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 6 actgtttttaaaggatgcac gtggacgtgg aatgtaaata tggaagacaa gagcagtgaa 60 aactggaataaagatattga gatcttgcag aatcgagctt ttgtgcttga ttattactat 120 tatctatgatattatattag ctatatgttg tctttgtacc gtagatgtgc tgtattgaac 180 ataaaaccgccgacatcgta taataaagag gaagggagaa aaggatttta gggacgaagg 240 ccgaaatgtaagagatctca ctcccaggac aagccaccta cgccgtcaca ccagtcccct 300 tctctcttcatcaagtaagt tatcccacag gatcctcagc tcctccatct gcctccaaca 360 tatagcccccagttcctgga atccctcctt cccctcagcc gtaagatcca ctgcaccacg 420 cttgcgccttcagaccctat cctccgccac aacagccggc cccaggaaca cccccatagc 480 cccatccgtcaacctccgaa gatacgacgg aggctacacc taatcgagac gccgaagacc 540 cccatttcaccctgccgccg ccgtgtttcc ggtacggtgc ttcggcggcg tcgagttctg 600 attgtgtggagagagcaagc cagacagggc tgcaagcgcc taggtaggtg gataaggtat 660 gccaaggggagccgaggatg cgggctagcc aaaatgcggc gaccattgcg tagatgagga 720 ttgtaggaaggggtataatc gccgtagcgc agattccggg gtgagagagg tatacgttgg 780 gtatggtcgaggcggtctca ggtccggtgc tgggcttgga atcggtttcg aagtcggtgg 840 aatagaagcttttcacccag ggagcagtac tgggaaggtc tgaggtgagg gataggaggt 900 ctgttagcgcttttgatgac tcgtagggag ctttggaccg gagcgcctga aaatcatcaa 960 catcgaagaagttgatcgtg gcttcagtgc tggagagcca tatcacgcgt ccgggtccgt 1020 tgggggatccggatcggtgc aggagaggca tgacattatg cgcgagcatg tagtggccga 1080 agacgttggcgcagaagacg gttcccaggc ggggctcctt gtcggtcaca gtaattgtct 1140 ggttgtccgttatgagacct acaggcgcaa ttttgtactt tggccacgtc gtcgcatgga 1200 taatgtcggtgcaaacgctc catacggcca gaggccaatt gaggccagac caacccccta 1260 tcccggcattaagcacaatc gcgtcgagtt ttgggaaggt cttgttcagg cgacgggata 1320 gcgcgcggaccgagaggagg ttgcagaggt cgacattttc aggaacgaag gtcactcgag 1380 cggtggccgaagcaccgaag gtggaggtgc ggaggtgatt ctgtaggttg cgaagggtgt 1440 cgcttccctttcttgtgctc cgggtagtga agatgattgt caatgaacga tggtcgttcc 1500 gatgagacgccaggaattca tctgcaagac ggcaacacgt tgagtatcct agtccgctaa 1560 acggtgaaaacccacaaggg gtaacagtta gaaacataaa ccactaacgg accgcatgag 1620 ataggagacagagcgcaggg tttcactgta cctgttggca ccggtcacga gcacaaagac 1680 tttctccttgaccccttcga tgccgtccgt aagcatggtg gctcaattgc gcttgtgccc 1740 ctgtgatcaagacttttg 1758 <210> SEQ ID NO 7 <211> LENGTH: 1792 <212> TYPE: DNA <213>ORGANISM: Aspergillus nidulans <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (230)...(309) <221> NAME/KEY: CDS <222> LOCATION:(375)...(815) <221> NAME/KEY: CDS <222> LOCATION: (876)...(1149) <221>NAME/KEY: CDS <222> LOCATION: (1200)...(1475) <400> SEQUENCE: 7gaattcctgt gatggagcag aacctcggag tatgctccga tgtcagtaca ttaaattttg 60tagcgatcca cgtgatttct attttgcgtc cgcaataggt cttctgatac ggctgaagaa 120atatagtacg tggtccagtg cctatagacg gaaagtattt tcgtacggtt ggctcccaag 180gcaataggtc aacctcgcat acggagaata acggtacggt cctgaagga atg agg gga 238Met Arg Gly 1 tgt att ctc ctt ctc cga ggg cca gaa ggg gaa cag gcc cgcact gat 286 Cys Ile Leu Leu Leu Arg Gly Pro Glu Gly Glu Gln Ala Arg ThrAsp 5 10 15 ccg gcg aaa att tcc cct ctc ga gtcttcgctc tcccccccacacggctgact 339 Pro Ala Lys Ile Ser Pro Leu Asp 20 25 aacccttccattcttgcccg catccagcca gccag c ctt ttg tcg ccg ccc ttg 393 Leu Leu SerPro Pro Leu 30 gtt cgg gct act gtc atc ttc cct tct tca tct tca tgc cgctct cga 441 Val Arg Ala Thr Val Ile Phe Pro Ser Ser Ser Ser Cys Arg SerArg 35 40 45 ctg aaa tat tca gtc tct tgc tct gat tta cag tta cta cgc gcagac 489 Leu Lys Tyr Ser Val Ser Cys Ser Asp Leu Gln Leu Leu Arg Ala Asp50 55 60 65 acg ctg cac atc tcc gcg atc atg acc gaa tcc act caa gaa cagggc 537 Thr Leu His Ile Ser Ala Ile Met Thr Glu Ser Thr Gln Glu Gln Gly70 75 80 aac gat ggc cag cga atg ccc ccc gcc ccg gcg acc ccc gtt gag gat585 Asn Asp Gly Gln Arg Met Pro Pro Ala Pro Ala Thr Pro Val Glu Asp 8590 95 tac gtc ttc cct gaa tat cgc ctg aag cgt gtg atg gat gac ccg gaa633 Tyr Val Phe Pro Glu Tyr Arg Leu Lys Arg Val Met Asp Asp Pro Glu 100105 110 aag acg ccg cta ttg ctt ata gct tgc ggt tca ttc tca cct att acg681 Lys Thr Pro Leu Leu Leu Ile Ala Cys Gly Ser Phe Ser Pro Ile Thr 115120 125 ttc ctg cac ctg cgc atg ttc gaa atg gcc gcc gat tac gtc aaa ctg729 Phe Leu His Leu Arg Met Phe Glu Met Ala Ala Asp Tyr Val Lys Leu 130135 140 145 agc aca gat ttc gaa ata att gga ggt tat ctt tcg ccc gtc tcggac 777 Ser Thr Asp Phe Glu Ile Ile Gly Gly Tyr Leu Ser Pro Val Ser Asp150 155 160 gcc tac cgc aag gca ggt ctt gcg agt gcc aat cac aggtagttactt 825 Ala Tyr Arg Lys Ala Gly Leu Ala Ser Ala Asn His Arg 165170 taacacactt cttccatagt tactatccag gactgatctg gcggctttag a att gca 882Ile Ala 175 atg tgc caa cga gcc gtg gac caa acg tca gac tgg atg atg gtggat 930 Met Cys Gln Arg Ala Val Asp Gln Thr Ser Asp Trp Met Met Val Asp180 185 190 aca tgg gag ccg atg cac aag gag tac cag cca act gcc atc gtactg 978 Thr Trp Glu Pro Met His Lys Glu Tyr Gln Pro Thr Ala Ile Val Leu195 200 205 gat cat ttt gac tac gag atc aac act gtc cgc aaa ggt atc gatacc 1026 Asp His Phe Asp Tyr Glu Ile Asn Thr Val Arg Lys Gly Ile Asp Thr210 215 220 gga aaa ggc act cga aag cga gtg caa gtc gtc tta ttg gcc ggggca 1074 Gly Lys Gly Thr Arg Lys Arg Val Gln Val Val Leu Leu Ala Gly Ala225 230 235 240 gat ttg gtc cat acc atg tct acg ccc gga gta tgg agt gagaag gat 1122 Asp Leu Val His Thr Met Ser Thr Pro Gly Val Trp Ser Glu LysAsp 245 250 255 ctc gat cat att ctt gga cag tac ggg gtatgttatgttgtatctat 1169 Leu Asp His Ile Leu Gly Gln Tyr Gly 260 265 cctaaacttcgcgcaagcta actggtctag act ttc atc gtc gag cga agc ggg 1223 Thr Phe IleVal Glu Arg Ser Gly 270 aca gat att gac gag gcg ctc gcg gca ttg cag ccatgg aaa aag aat 1271 Thr Asp Ile Asp Glu Ala Leu Ala Ala Leu Gln Pro TrpLys Lys Asn 275 280 285 atc cat gtt att caa caa ctt att caa aat gac gttagc agc act aag 1319 Ile His Val Ile Gln Gln Leu Ile Gln Asn Asp Val SerSer Thr Lys 290 295 300 305 att cgc tta ttc ctc agg cga gat atg agc gtacgc tac ttg atc cct 1367 Ile Arg Leu Phe Leu Arg Arg Asp Met Ser Val ArgTyr Leu Ile Pro 310 315 320 gac ccg gtg att gag tac atc tat gag aat aacctc tac atg gac gac 1415 Asp Pro Val Ile Glu Tyr Ile Tyr Glu Asn Asn LeuTyr Met Asp Asp 325 330 335 ggt acg aca caa ccg acg gcc gac aag ggc aagaca cga gag gag ccc 1463 Gly Thr Thr Gln Pro Thr Ala Asp Lys Gly Lys ThrArg Glu Glu Pro 340 345 350 gcg cct tca aat tagcattgct caaaaagccagataaggcca cgcgacgacg 1515 Ala Pro Ser Asn 355 tcatgacgac cattgctggtttcacgaaga tatcaaaccg ccgggcgaat gcaatctctg 1575 cgctgatctg agcaagcactgattccggta agccgcaagt tgggggagga tttaatgagc 1635 ccaaccgtat gggtttgttccggtcaagtc actgcgatta acgacacgcc ttatgactgt 1695 catatcgaca ggtccctctccagagccggc ctacacaaca gtgatgctgg cgttcttcta 1755 ttccaagccc tcaacatctaagtgcagcgg cgaattc 1792 <210> SEQ ID NO 8 <211> LENGTH: 27 <212> TYPE:PRT <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 8 Met Arg GlyCys Ile Leu Leu Leu Arg Gly Pro Glu Gly Glu Gln Ala 1 5 10 15 Arg ThrAsp Pro Ala Lys Ile Ser Pro Leu Asp 20 25 <210> SEQ ID NO 9 <211>LENGTH: 1792 <212> TYPE: DNA <213> ORGANISM: Aspergillus nidulans <400>SEQUENCE: 9 gaattcgccg ctgcacttag atgttgaggg cttggaatag aagaacgccagcatcactgt 60 tgtgtaggcc ggctctggag agggacctgt cgatatgaca gtcataaggcgtgtcgttaa 120 tcgcagtgac ttgaccggaa caaacccata cggttgggct cattaaatcctcccccaact 180 tgcggcttac cggaatcagt gcttgctcag atcagcgcag agattgcattcgcccggcgg 240 tttgatatct tcgtgaaacc agcaatggtc gtcatgacgt cgtcgcgtggccttatctgg 300 ctttttgagc aatgctaatt tgaaggcgcg ggctcctctc gtgtcttgcccttgtcggcc 360 gtcggttgtg tcgtaccgtc gtccatgtag aggttattct catagatgtactcaatcacc 420 gggtcaggga tcaagtagcg tacgctcata tctcgcctga ggaataagcgaatcttagtg 480 ctgctaacgt cattttgaat aagttgttga ataacatgga tattctttttccatggctgc 540 aatgccgcga gcgcctcgtc aatatctgtc ccgcttcgct cgacgatgaaagtctagacc 600 agttagcttg cgcgaagttt aggatagata caacataaca taccccgtactgtccaagaa 660 tatgatcgag atccttctca ctccatactc cgggcgtaga catggtatggaccaaatctg 720 ccccggccaa taagacgact tgcactcgct ttcgagtgcc ttttccggtatcgatacctt 780 tgcggacagt gttgatctcg tagtcaaaat gatccagtac gatggcagttggctggtact 840 ccttgtgcat cggctcccat gtatccacca tcatccagtc tgacgtttggtccacggctc 900 gttggcacat tgcaattcta aagccgccag atcagtcctg gatagtaactatggaagaag 960 tgtgttaaag taactacctg tgattggcac tcgcaagacc tgccttgcggtaggcgtccg 1020 agacgggcga aagataacct ccaattattt cgaaatctgt gctcagtttgacgtaatcgg 1080 cggccatttc gaacatgcgc aggtgcagga acgtaatagg tgagaatgaaccgcaagcta 1140 taagcaatag cggcgtcttt tccgggtcat ccatcacacg cttcaggcgatattcaggga 1200 agacgtaatc ctcaacgggg gtcgccgggg cggggggcat tcgctggccatcgttgccct 1260 gttcttgagt ggattcggtc atgatcgcgg agatgtgcag cgtgtctgcgcgtagtaact 1320 gtaaatcaga gcaagagact gaatatttca gtcgagagcg gcatgaagatgaagaaggga 1380 agatgacagt agcccgaacc aagggcggcg acaaaaggct ggctggctggatgcgggcaa 1440 gaatggaagg gttagtcagc cgtgtggggg ggagagcgaa gactcgagaggggaaatttt 1500 cgccggatca gtgcgggcct gttccccttc tggccctcgg agaaggagaatacatcccct 1560 cattccttca ggaccgtacc gttattctcc gtatgcgagg ttgacctattgccttgggag 1620 ccaaccgtac gaaaatactt tccgtctata ggcactggac cacgtactatatttcttcag 1680 ccgtatcaga agacctattg cggacgcaaa atagaaatca cgtggatcgctacaaaattt 1740 aatgtactga catcggagca tactccgagg ttctgctcca tcacaggaattc 1792 <210> SEQ ID NO 10 <211> LENGTH: 1899 <212> TYPE: DNA <213>ORGANISM: Aspergillus nidulans <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (453)...(627) <221> NAME/KEY: CDS <222> LOCATION:(686)...(890) <221> NAME/KEY: CDS <222> LOCATION: (949)...(1157) <221>NAME/KEY: CDS <222> LOCATION: (1212)...(1288) <400> SEQUENCE: 10ttgccttctt agacttgata tctgaaggaa tataacggaa gagatcatct ggtttgatgg 60tactgtatta gcgggagcac gtgattattt ccctccgata ggccagtggc gtatgtcata 120aggaagactg acgcctggag gggaaaacac ctccctcgcc cgagttccat cttatcactt 180tcacgctcga tctctccaag tttctggctt cattgactga gtcgctcgcc ttgcctagtg 240ggtagattta gatctagtcg caaatcactt gcctacattc tcgaacctgt ttgttcagcc 300ttgcggttcc cctcactact tatctcttct taccttctac cgtttcgaaa acacttcctc 360ctgcggcgag actagtatct atcgcctgtc gcccactttc accaccgtgt ttcactagga 420gaatagtgaa agactcaagt cgtctaccaa aa atg tgg tca tgg ttc cgg tgg 473 MetTrp Ser Trp Phe Arg Trp 1 5 tgc ggc cgc gca gaa gcg caa gga agc gcc gaaaac gca atc ctc cag 521 Cys Gly Arg Ala Glu Ala Gln Gly Ser Ala Glu AsnAla Ile Leu Gln 10 15 20 ctt cga agc cac ctt gac atg cta cag aag cga gaaaag cac cta gaa 569 Leu Arg Ser His Leu Asp Met Leu Gln Lys Arg Glu LysHis Leu Glu 25 30 35 aac caa atg aac gaa caa gag gcc atc gct aaa aag aacgtg acc acg 617 Asn Gln Met Asn Glu Gln Glu Ala Ile Ala Lys Lys Asn ValThr Thr 40 45 50 55 aat aag aac g gtgtgtatat tatgggacct ttatacaagttcccatgctg 667 Asn Lys Asn atttgaccac caccgcag cc gcc aaa gcc gcg ctccga cgg aaa aag gtg 717 Ala Ala Lys Ala Ala Leu Arg Arg Lys Lys Val 6065 cac gag aag aac tta gaa cag acg cag gct cag att gta cag ctt gag 765His Glu Lys Asn Leu Glu Gln Thr Gln Ala Gln Ile Val Gln Leu Glu 70 75 8085 cag cag ata tac tct att gaa gcc gcc aat att aac cac gag acc ctg 813Gln Gln Ile Tyr Ser Ile Glu Ala Ala Asn Ile Asn His Glu Thr Leu 90 95100 gcc gcc atg aag gcc gcc ggt gca gct atg gag aag att cac aac ggc 861Ala Ala Met Lys Ala Ala Gly Ala Ala Met Glu Lys Ile His Asn Gly 105 110115 atg acc gtc gaa cag gtc gac gag aca at gtacgtccct tactgtaccg 910 MetThr Val Glu Gln Val Asp Glu Thr Met 120 125 ctggtgacat accggaattggcatgctaac agactcag g gac aaa ctg cgg gaa 964 Asp Lys Leu Arg Glu 130caa caa gcc atc aac gac gaa atc gcg att gcc atc aca aac ccg ggg 1012 GlnGln Ala Ile Asn Asp Glu Ile Ala Ile Ala Ile Thr Asn Pro Gly 135 140 145ttc ggc gag cag gtg gac gaa gaa gat ctg gag gcg gaa ctc gag ggc 1060 PheGly Glu Gln Val Asp Glu Glu Asp Leu Glu Ala Glu Leu Glu Gly 150 155 160atg gag cag gag gct atg gac gag cgc atg ctc cac aca ggc aca gta 1108 MetGlu Gln Glu Ala Met Asp Glu Arg Met Leu His Thr Gly Thr Val 165 170 175180 cca gtt gca gat cag ctc aat cgg cta cct gcg cca gcg aat gca gaa 1156Pro Val Ala Asp Gln Leu Asn Arg Leu Pro Ala Pro Ala Asn Ala Glu 185 190195 c gtaaggctct ccctttccca cctcaaaagc gaactccgac tgacagcctt 1207 ccagcc gcc aaa gcg aaa cag aaa gca gaa gaa gaa gac gag gaa gcc 1255 Pro AlaLys Ala Lys Gln Lys Ala Glu Glu Glu Asp Glu Glu Ala 200 205 210 gag ttggag aag tta cgc gcg gaa atg gcc atg tgagagtggt cctggtgctt 1308 Glu LeuGlu Lys Leu Arg Ala Glu Met Ala Met 215 220 tggtctcttt ggtctaactttaatcttttt tcttccccct acacatatga tgaacaggga 1368 atcgttatca tgacgcactacgattagcca agcactgtgt tctttttccg tcggctcgtt 1428 gcgattcctt cttctccgcggcgtaattac ttatctagtt gtaccaacta ccccgcgagg 1488 cttctgttga ggcgagagcgaaagcccaga cgtgtcgccc ttgccctgat tactggccac 1548 tcccgtccga gcacgctacctccgttctgt ccacgctgtg tatcccactc tgtaataatc 1608 taccaagtga atacttttctggatgatttg aagggcctat gtttcctacg ccatcatgtc 1668 attagatatg ttttgtggatcatgtttccc cagcgcaatt gatgcccatt tgcagttcac 1728 actcgtgtca tatgaacctcagaatatgaa agccgcttct caacccagca aaacgtcact 1788 gaggattaaa attgagtaattgagtaaaac taaattagta gctagataac tcccgtttcc 1848 caccagacct aacaccgtccaaacagataa tcaacaagga aaagaaagaa a 1899 <210> SEQ ID NO 11 <211> LENGTH:59 <212> TYPE: PRT <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE:11 Met Trp Ser Trp Phe Arg Trp Cys Gly Arg Ala Glu Ala Gln Gly Ser 1 510 15 Ala Glu Asn Ala Ile Leu Gln Leu Arg Ser His Leu Asp Met Leu Gln 2025 30 Lys Arg Glu Lys His Leu Glu Asn Gln Met Asn Glu Gln Glu Ala Ile 3540 45 Ala Lys Lys Asn Val Thr Thr Asn Lys Asn Ala 50 55 <210> SEQ ID NO12 <211> LENGTH: 1899 <212> TYPE: DNA <213> ORGANISM: Aspergillusnidulans <400> SEQUENCE: 12 tttctttctt ttccttgttg attatctgtt tggacggtgttaggtctggt gggaaacggg 60 agttatctag ctactaattt agttttactc aattactcaattttaatcct cagtgacgtt 120 ttgctgggtt gagaagcggc tttcatattc tgaggttcatatgacacgag tgtgaactgc 180 aaatgggcat caattgcgct ggggaaacat gatccacaaaacatatctaa tgacatgatg 240 gcgtaggaaa cataggccct tcaaatcatc cagaaaagtattcacttggt agattattac 300 agagtgggat acacagcgtg gacagaacgg aggtagcgtgctcggacggg agtggccagt 360 aatcagggca agggcgacac gtctgggctt tcgctctcgcctcaacagaa gcctcgcggg 420 gtagttggta caactagata agtaattacg ccgcggagaagaaggaatcg caacgagccg 480 acggaaaaag aacacagtgc ttggctaatc gtagtgcgtcatgataacga ttccctgttc 540 atcatatgtg tagggggaag aaaaaagatt aaagttagaccaaagagacc aaagcaccag 600 gaccactctc acatggccat ttccgcgcgt aacttctccaactcggcttc ctcgtcttct 660 tcttctgctt tctgtttcgc tttggcggct ggaaggctgtcagtcggagt tcgcttttga 720 ggtgggaaag ggagagcctt acgttctgca ttcgctggcgcaggtagccg attgagctga 780 tctgcaactg gtactgtgcc tgtgtggagc atgcgctcgtccatagcctc ctgctccatg 840 ccctcgagtt ccgcctccag atcttcttcg tccacctgctcgccgaaccc cgggtttgtg 900 atggcaatcg cgatttcgtc gttgatggct tgttgttcccgcagtttgtc cctgagtctg 960 ttagcatgcc aattccggta tgtcaccagc ggtacagtaagggacgtaca ttgtctcgtc 1020 gacctgttcg acggtcatgc cgttgtgaat cttctccatagctgcaccgg cggccttcat 1080 ggcggccagg gtctcgtggt taatattggc ggcttcaatagagtatatct gctgctcaag 1140 ctgtacaatc tgagcctgcg tctgttctaa gttcttctcgtgcacctttt tccgtcggag 1200 cgcggctttg gcggctgcgg tggtggtcaa atcagcatgggaacttgtat aaaggtccca 1260 taatatacac accgttctta ttcgtggtca cgttctttttagcgatggcc tcttgttcgt 1320 tcatttggtt ttctaggtgc ttttctcgct tctgtagcatgtcaaggtgg cttcgaagct 1380 ggaggattgc gttttcggcg cttccttgcg cttctgcgcggccgcaccac cggaaccatg 1440 accacatttt tggtagacga cttgagtctt tcactattctcctagtgaaa cacggtggtg 1500 aaagtgggcg acaggcgata gatactagtc tcgccgcaggaggaagtgtt ttcgaaacgg 1560 tagaaggtaa gaagagataa gtagtgaggg gaaccgcaaggctgaacaaa caggttcgag 1620 aatgtaggca agtgatttgc gactagatct aaatctacccactaggcaag gcgagcgact 1680 cagtcaatga agccagaaac ttggagagat cgagcgtgaaagtgataaga tggaactcgg 1740 gcgagggagg tgttttcccc tccaggcgtc agtcttccttatgacatacg ccactggcct 1800 atcggaggga aataatcacg tgctcccgct aatacagtaccatcaaacca gatgatctct 1860 tccgttatat tccttcagat atcaagtcta agaaggcaa1899 <210> SEQ ID NO 13 <211> LENGTH: 3800 <212> TYPE: DNA <213>ORGANISM: Saccharomyces cerevisiae <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (306)...(3458) <400> SEQUENCE: 13 tttttcttgt cagtctgaaaatttttcatt tggttttttg aaaaaaatcc tgcctaatat 60 ggtatcaaga ggaataacaagaaaaaaaaa tcatggggga tacaaaggaa aacaaggaga 120 taatgcaggt tatactgaattgctcatagt attagcctaa agcactttac ctctgattta 180 ttgcatctat cgtattcttgagttattgcg acttttaaaa tccgtgcacc gcatatgaaa 240 gggtagagcc ttcgtgtttgtttacctttt tagctctttg aagatcaaac aaaaacactt 300 cagta atg cct aca gccttg gat aag aca aag aag tta aca gcc gcg ccc 350 Met Pro Thr Ala Leu AspLys Thr Lys Lys Leu Thr Ala Ala Pro 1 5 10 15 atc atg caa gat cct gatggt att gac att aat acg aaa atc ttt aac 398 Ile Met Gln Asp Pro Asp GlyIle Asp Ile Asn Thr Lys Ile Phe Asn 20 25 30 tca gtt gct gaa gta ttt caaaag gca cag ggt tct tat gca gga cac 446 Ser Val Ala Glu Val Phe Gln LysAla Gln Gly Ser Tyr Ala Gly His 35 40 45 agg aag cat ata gca gtt ttg aagaaa att cag tca aag gct gtt gag 494 Arg Lys His Ile Ala Val Leu Lys LysIle Gln Ser Lys Ala Val Glu 50 55 60 caa ggc tat gaa gat gct ttt aac ttttgg ttc gat aaa tta gtt act 542 Gln Gly Tyr Glu Asp Ala Phe Asn Phe TrpPhe Asp Lys Leu Val Thr 65 70 75 aag atc ctt cct ctg aaa aag aat gag attatc gga gac agg ata gta 590 Lys Ile Leu Pro Leu Lys Lys Asn Glu Ile IleGly Asp Arg Ile Val 80 85 90 95 aag tta gta gct gca ttt ata gct tct ttagaa agg gag ttg ata ttg 638 Lys Leu Val Ala Ala Phe Ile Ala Ser Leu GluArg Glu Leu Ile Leu 100 105 110 gcc aaa aaa caa aac tat aag ctc acg aatgat gaa gaa ggg ata ttc 686 Ala Lys Lys Gln Asn Tyr Lys Leu Thr Asn AspGlu Glu Gly Ile Phe 115 120 125 tca agg ttc gtc gat cag ttc ata aga catgtt ttg cgt ggt gtg gaa 734 Ser Arg Phe Val Asp Gln Phe Ile Arg His ValLeu Arg Gly Val Glu 130 135 140 agc cct gac aag aac gtc aga ttt aga gtttta cag tta tta gcc gtt 782 Ser Pro Asp Lys Asn Val Arg Phe Arg Val LeuGln Leu Leu Ala Val 145 150 155 ata atg gat aat ata ggg gaa atc gat gaatca ctt ttc aat tta tta 830 Ile Met Asp Asn Ile Gly Glu Ile Asp Glu SerLeu Phe Asn Leu Leu 160 165 170 175 ata ttg tct tta aat aag agg att tatgat aga gaa cca acg gtt agg 878 Ile Leu Ser Leu Asn Lys Arg Ile Tyr AspArg Glu Pro Thr Val Arg 180 185 190 ata cag gct gtg ttt tgt tta act aaattt cag gat gaa gag caa act 926 Ile Gln Ala Val Phe Cys Leu Thr Lys PheGln Asp Glu Glu Gln Thr 195 200 205 gaa cat tta act gag ctt tct gat aatgaa gaa aat ttt gaa gct acg 974 Glu His Leu Thr Glu Leu Ser Asp Asn GluGlu Asn Phe Glu Ala Thr 210 215 220 aga act cta gtt gct tct atc cag aacgat ccg tca gct gaa gta cgg 1022 Arg Thr Leu Val Ala Ser Ile Gln Asn AspPro Ser Ala Glu Val Arg 225 230 235 agg gct gca atg ctg aat ttg atc aatgat aat aat act aga ccg tat 1070 Arg Ala Ala Met Leu Asn Leu Ile Asn AspAsn Asn Thr Arg Pro Tyr 240 245 250 255 atc ttg gag agg gct aga gat gtaaac atc gtt aat aga agg ctc gtg 1118 Ile Leu Glu Arg Ala Arg Asp Val AsnIle Val Asn Arg Arg Leu Val 260 265 270 tat tcg aga att ttg aaa tca atggga aga aag tgt ttc gat gat att 1166 Tyr Ser Arg Ile Leu Lys Ser Met GlyArg Lys Cys Phe Asp Asp Ile 275 280 285 gag ccg cat att ttt gat caa ttgatt gag tgg ggt tta gaa gat agg 1214 Glu Pro His Ile Phe Asp Gln Leu IleGlu Trp Gly Leu Glu Asp Arg 290 295 300 gaa tta tca gtg aga aat gcg tgtaag aga ctc att gct cat gat tgg 1262 Glu Leu Ser Val Arg Asn Ala Cys LysArg Leu Ile Ala His Asp Trp 305 310 315 tta aat gct ctg gat ggc gat ttgata gaa tta cta gaa aaa ttg gat 1310 Leu Asn Ala Leu Asp Gly Asp Leu IleGlu Leu Leu Glu Lys Leu Asp 320 325 330 335 gtc tca aga tcc tca gtg tgtgtt aag gct ata gaa gca ctt ttt caa 1358 Val Ser Arg Ser Ser Val Cys ValLys Ala Ile Glu Ala Leu Phe Gln 340 345 350 tca agg cca gat ata tta tctaaa atc aaa ttt cct gaa agt att tgg 1406 Ser Arg Pro Asp Ile Leu Ser LysIle Lys Phe Pro Glu Ser Ile Trp 355 360 365 aaa gac ttt acc gta gaa attgcc ttc ctc ttt cgg gct att tat ttg 1454 Lys Asp Phe Thr Val Glu Ile AlaPhe Leu Phe Arg Ala Ile Tyr Leu 370 375 380 tac tgt ttg gat aat aat ataaca gaa atg ctg gaa gaa aac ttt cca 1502 Tyr Cys Leu Asp Asn Asn Ile ThrGlu Met Leu Glu Glu Asn Phe Pro 385 390 395 gaa gcc tca aaa tta tcc gagcat tta aac cat tat att ctt ctc aga 1550 Glu Ala Ser Lys Leu Ser Glu HisLeu Asn His Tyr Ile Leu Leu Arg 400 405 410 415 tat cat cac aac gac atttct aat gac tct cag tcg cat ttt gat tat 1598 Tyr His His Asn Asp Ile SerAsn Asp Ser Gln Ser His Phe Asp Tyr 420 425 430 aac act tta gag ttt attatt gag caa cta tcg att gcc gcc gaa agg 1646 Asn Thr Leu Glu Phe Ile IleGlu Gln Leu Ser Ile Ala Ala Glu Arg 435 440 445 tat gat tat agc gat gaggtt gga agg aga tcg atg ctt aca gtg gta 1694 Tyr Asp Tyr Ser Asp Glu ValGly Arg Arg Ser Met Leu Thr Val Val 450 455 460 cga aat atg ctg gcc ttaact aca ctc tcc gaa cct ctt att aaa att 1742 Arg Asn Met Leu Ala Leu ThrThr Leu Ser Glu Pro Leu Ile Lys Ile 465 470 475 ggt att cgt gta atg aaaagt ctg tcc ata aat gaa aaa gat ttt gta 1790 Gly Ile Arg Val Met Lys SerLeu Ser Ile Asn Glu Lys Asp Phe Val 480 485 490 495 aca atg gca ata gaaatc att aat gat att aga gac gac gat att gaa 1838 Thr Met Ala Ile Glu IleIle Asn Asp Ile Arg Asp Asp Asp Ile Glu 500 505 510 aaa caa gaa caa gaagag aaa ata aaa agc aag aag att aat cgc aga 1886 Lys Gln Glu Gln Glu GluLys Ile Lys Ser Lys Lys Ile Asn Arg Arg 515 520 525 aat gag act tcc gtcgat gaa gag gac gaa aac ggc aca cat aat gac 1934 Asn Glu Thr Ser Val AspGlu Glu Asp Glu Asn Gly Thr His Asn Asp 530 535 540 gaa gtt aac gag gatgaa gaa gac gac aat att tca tcc ttc cat tct 1982 Glu Val Asn Glu Asp GluGlu Asp Asp Asn Ile Ser Ser Phe His Ser 545 550 555 gct gta gaa aac ttagtg cag gga aac ggc aac gta tct gag agt gac 2030 Ala Val Glu Asn Leu ValGln Gly Asn Gly Asn Val Ser Glu Ser Asp 560 565 570 575 ata ata aat aatctc cca ccc gaa aag gaa gcg tcc tca gca aca att 2078 Ile Ile Asn Asn LeuPro Pro Glu Lys Glu Ala Ser Ser Ala Thr Ile 580 585 590 gtt ctc tgt cttaca agg tca tca tat atg cta gaa cta gtt aac aca 2126 Val Leu Cys Leu ThrArg Ser Ser Tyr Met Leu Glu Leu Val Asn Thr 595 600 605 ccg tta aca gaaaac att tta att gcg tcg ttg atg gac act ttg atc 2174 Pro Leu Thr Glu AsnIle Leu Ile Ala Ser Leu Met Asp Thr Leu Ile 610 615 620 aca cca gcg gttaga aat acc gcg cca aat att agg gag ctt ggt gtc 2222 Thr Pro Ala Val ArgAsn Thr Ala Pro Asn Ile Arg Glu Leu Gly Val 625 630 635 aag aac ctt ggttta tgt tgt ctc ttg gat gtg aag ttg gct att gat 2270 Lys Asn Leu Gly LeuCys Cys Leu Leu Asp Val Lys Leu Ala Ile Asp 640 645 650 655 aac atg tacatc tta ggt atg tgc gtt tcg aaa ggt aat gca tca tta 2318 Asn Met Tyr IleLeu Gly Met Cys Val Ser Lys Gly Asn Ala Ser Leu 660 665 670 aag tat attgcg tta caa gtc att gta gat att ttt tcc gta cat ggg 2366 Lys Tyr Ile AlaLeu Gln Val Ile Val Asp Ile Phe Ser Val His Gly 675 680 685 aac act gtggta gac gga gaa ggc aaa gtt gac tca atc tcg ttg cac 2414 Asn Thr Val ValAsp Gly Glu Gly Lys Val Asp Ser Ile Ser Leu His 690 695 700 aaa ata ttttac aag gtt tta aag aat aac ggt tta ccg gaa tgt cag 2462 Lys Ile Phe TyrLys Val Leu Lys Asn Asn Gly Leu Pro Glu Cys Gln 705 710 715 gtg ata gcagcg gag ggt tta tgc aaa cta ttt ttg gca gac gtg ttc 2510 Val Ile Ala AlaGlu Gly Leu Cys Lys Leu Phe Leu Ala Asp Val Phe 720 725 730 735 act gatgat gat ttg ttt gaa acg ttg gtt ttg tca tat ttt tcg ccg 2558 Thr Asp AspAsp Leu Phe Glu Thr Leu Val Leu Ser Tyr Phe Ser Pro 740 745 750 ata aattcc tca aac gaa gcg ctg gta cag gca ttt gcc ttc tgc att 2606 Ile Asn SerSer Asn Glu Ala Leu Val Gln Ala Phe Ala Phe Cys Ile 755 760 765 cca gtctat tgt ttt tca cat cct gct cat caa caa cgt atg tct agg 2654 Pro Val TyrCys Phe Ser His Pro Ala His Gln Gln Arg Met Ser Arg 770 775 780 acg gctgcg gac ata ctc tta aga cta tgt gtt ctt tgg gac gat tta 2702 Thr Ala AlaAsp Ile Leu Leu Arg Leu Cys Val Leu Trp Asp Asp Leu 785 790 795 cag agctct gta ata cct gag gta gac cgt gaa gct atg cta aag cct 2750 Gln Ser SerVal Ile Pro Glu Val Asp Arg Glu Ala Met Leu Lys Pro 800 805 810 815 aacata ata ttt caa cag ttg cta ttt tgg act gat cca cgt aac tta 2798 Asn IleIle Phe Gln Gln Leu Leu Phe Trp Thr Asp Pro Arg Asn Leu 820 825 830 gttaac cag aca ggt tca aca aaa aaa gat aca gtg cag ctt aca ttc 2846 Val AsnGln Thr Gly Ser Thr Lys Lys Asp Thr Val Gln Leu Thr Phe 835 840 845 ttgatc gat gtg ctc aaa ata tac gct caa att gag aag aaa gaa ata 2894 Leu IleAsp Val Leu Lys Ile Tyr Ala Gln Ile Glu Lys Lys Glu Ile 850 855 860 aagaag atg atc atc act aat ata aac gct ata ttt ctt tct tct gaa 2942 Lys LysMet Ile Ile Thr Asn Ile Asn Ala Ile Phe Leu Ser Ser Glu 865 870 875 caagat tat tct act ttg aaa gaa ctt ctt gag tat tct gac gat att 2990 Gln AspTyr Ser Thr Leu Lys Glu Leu Leu Glu Tyr Ser Asp Asp Ile 880 885 890 895gca gaa aat gat aat tta gac aat gtt agc aaa aat gct ctg gac aag 3038 AlaGlu Asn Asp Asn Leu Asp Asn Val Ser Lys Asn Ala Leu Asp Lys 900 905 910cta agg aat aat ttg aat tcg ctg att gaa gag atc aat gaa agg tca 3086 LeuArg Asn Asn Leu Asn Ser Leu Ile Glu Glu Ile Asn Glu Arg Ser 915 920 925gaa act cag aca aaa gat gag aac aac act gcg aat gac caa tac tcg 3134 GluThr Gln Thr Lys Asp Glu Asn Asn Thr Ala Asn Asp Gln Tyr Ser 930 935 940tct att ttg ggg aat tca ttc aat aaa tct tca aat gac acc ata gaa 3182 SerIle Leu Gly Asn Ser Phe Asn Lys Ser Ser Asn Asp Thr Ile Glu 945 950 955cac gct gct gat ata act gat gga aat aac aca gaa ttg act aaa aca 3230 HisAla Ala Asp Ile Thr Asp Gly Asn Asn Thr Glu Leu Thr Lys Thr 960 965 970975 act gtt aat att tcg gca gtt gac aat aca aca gag caa agt aac tca 3278Thr Val Asn Ile Ser Ala Val Asp Asn Thr Thr Glu Gln Ser Asn Ser 980 985990 agg aaa aga acg aga tca gaa gcg gag caa att gac aca tcc aaa aac 3326Arg Lys Arg Thr Arg Ser Glu Ala Glu Gln Ile Asp Thr Ser Lys Asn 995 10001005 ctg gaa aac atg agt att caa gac acg tca act gta gca aaa aat gta3374 Leu Glu Asn Met Ser Ile Gln Asp Thr Ser Thr Val Ala Lys Asn Val1010 1015 1020 agt ttt gtt tta cct gac gag aaa tca gat gca atg tcc atagat gaa 3422 Ser Phe Val Leu Pro Asp Glu Lys Ser Asp Ala Met Ser Ile AspGlu 1025 1030 1035 gaa gat aag gat tca gag tct ttc agc gag gtc tgttaaaattgat 3468 Glu Asp Lys Asp Ser Glu Ser Phe Ser Glu Val Cys 10401045 1050 atgcgagctc ttcatctatt taagttgatt ttttggttgt aaacatatttgtattttatt 3528 cttaggtttg ttaattcttc tacgcttacc agatatagat gctatatgttattgcattac 3588 gcacattacc cggtgggaca aattatggaa atattccaag gctataaattctttggtgaa 3648 aggaactgaa attatgtcca gtaatgcacc agaaatggac atataaaactattaatgcat 3708 tttattacaa ttatcctaag aaaatatcct atatataatt aaagtaaaagaaataagatc 3768 aaaagaacaa aataaagtcg agtagaattt tc 3800 <210> SEQ ID NO14 <211> LENGTH: 1051 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 14 Met Pro Thr Ala Leu Asp Lys Thr Lys LysLeu Thr Ala Ala Pro Ile 1 5 10 15 Met Gln Asp Pro Asp Gly Ile Asp IleAsn Thr Lys Ile Phe Asn Ser 20 25 30 Val Ala Glu Val Phe Gln Lys Ala GlnGly Ser Tyr Ala Gly His Arg 35 40 45 Lys His Ile Ala Val Leu Lys Lys IleGln Ser Lys Ala Val Glu Gln 50 55 60 Gly Tyr Glu Asp Ala Phe Asn Phe TrpPhe Asp Lys Leu Val Thr Lys 65 70 75 80 Ile Leu Pro Leu Lys Lys Asn GluIle Ile Gly Asp Arg Ile Val Lys 85 90 95 Leu Val Ala Ala Phe Ile Ala SerLeu Glu Arg Glu Leu Ile Leu Ala 100 105 110 Lys Lys Gln Asn Tyr Lys LeuThr Asn Asp Glu Glu Gly Ile Phe Ser 115 120 125 Arg Phe Val Asp Gln PheIle Arg His Val Leu Arg Gly Val Glu Ser 130 135 140 Pro Asp Lys Asn ValArg Phe Arg Val Leu Gln Leu Leu Ala Val Ile 145 150 155 160 Met Asp AsnIle Gly Glu Ile Asp Glu Ser Leu Phe Asn Leu Leu Ile 165 170 175 Leu SerLeu Asn Lys Arg Ile Tyr Asp Arg Glu Pro Thr Val Arg Ile 180 185 190 GlnAla Val Phe Cys Leu Thr Lys Phe Gln Asp Glu Glu Gln Thr Glu 195 200 205His Leu Thr Glu Leu Ser Asp Asn Glu Glu Asn Phe Glu Ala Thr Arg 210 215220 Thr Leu Val Ala Ser Ile Gln Asn Asp Pro Ser Ala Glu Val Arg Arg 225230 235 240 Ala Ala Met Leu Asn Leu Ile Asn Asp Asn Asn Thr Arg Pro TyrIle 245 250 255 Leu Glu Arg Ala Arg Asp Val Asn Ile Val Asn Arg Arg LeuVal Tyr 260 265 270 Ser Arg Ile Leu Lys Ser Met Gly Arg Lys Cys Phe AspAsp Ile Glu 275 280 285 Pro His Ile Phe Asp Gln Leu Ile Glu Trp Gly LeuGlu Asp Arg Glu 290 295 300 Leu Ser Val Arg Asn Ala Cys Lys Arg Leu IleAla His Asp Trp Leu 305 310 315 320 Asn Ala Leu Asp Gly Asp Leu Ile GluLeu Leu Glu Lys Leu Asp Val 325 330 335 Ser Arg Ser Ser Val Cys Val LysAla Ile Glu Ala Leu Phe Gln Ser 340 345 350 Arg Pro Asp Ile Leu Ser LysIle Lys Phe Pro Glu Ser Ile Trp Lys 355 360 365 Asp Phe Thr Val Glu IleAla Phe Leu Phe Arg Ala Ile Tyr Leu Tyr 370 375 380 Cys Leu Asp Asn AsnIle Thr Glu Met Leu Glu Glu Asn Phe Pro Glu 385 390 395 400 Ala Ser LysLeu Ser Glu His Leu Asn His Tyr Ile Leu Leu Arg Tyr 405 410 415 His HisAsn Asp Ile Ser Asn Asp Ser Gln Ser His Phe Asp Tyr Asn 420 425 430 ThrLeu Glu Phe Ile Ile Glu Gln Leu Ser Ile Ala Ala Glu Arg Tyr 435 440 445Asp Tyr Ser Asp Glu Val Gly Arg Arg Ser Met Leu Thr Val Val Arg 450 455460 Asn Met Leu Ala Leu Thr Thr Leu Ser Glu Pro Leu Ile Lys Ile Gly 465470 475 480 Ile Arg Val Met Lys Ser Leu Ser Ile Asn Glu Lys Asp Phe ValThr 485 490 495 Met Ala Ile Glu Ile Ile Asn Asp Ile Arg Asp Asp Asp IleGlu Lys 500 505 510 Gln Glu Gln Glu Glu Lys Ile Lys Ser Lys Lys Ile AsnArg Arg Asn 515 520 525 Glu Thr Ser Val Asp Glu Glu Asp Glu Asn Gly ThrHis Asn Asp Glu 530 535 540 Val Asn Glu Asp Glu Glu Asp Asp Asn Ile SerSer Phe His Ser Ala 545 550 555 560 Val Glu Asn Leu Val Gln Gly Asn GlyAsn Val Ser Glu Ser Asp Ile 565 570 575 Ile Asn Asn Leu Pro Pro Glu LysGlu Ala Ser Ser Ala Thr Ile Val 580 585 590 Leu Cys Leu Thr Arg Ser SerTyr Met Leu Glu Leu Val Asn Thr Pro 595 600 605 Leu Thr Glu Asn Ile LeuIle Ala Ser Leu Met Asp Thr Leu Ile Thr 610 615 620 Pro Ala Val Arg AsnThr Ala Pro Asn Ile Arg Glu Leu Gly Val Lys 625 630 635 640 Asn Leu GlyLeu Cys Cys Leu Leu Asp Val Lys Leu Ala Ile Asp Asn 645 650 655 Met TyrIle Leu Gly Met Cys Val Ser Lys Gly Asn Ala Ser Leu Lys 660 665 670 TyrIle Ala Leu Gln Val Ile Val Asp Ile Phe Ser Val His Gly Asn 675 680 685Thr Val Val Asp Gly Glu Gly Lys Val Asp Ser Ile Ser Leu His Lys 690 695700 Ile Phe Tyr Lys Val Leu Lys Asn Asn Gly Leu Pro Glu Cys Gln Val 705710 715 720 Ile Ala Ala Glu Gly Leu Cys Lys Leu Phe Leu Ala Asp Val PheThr 725 730 735 Asp Asp Asp Leu Phe Glu Thr Leu Val Leu Ser Tyr Phe SerPro Ile 740 745 750 Asn Ser Ser Asn Glu Ala Leu Val Gln Ala Phe Ala PheCys Ile Pro 755 760 765 Val Tyr Cys Phe Ser His Pro Ala His Gln Gln ArgMet Ser Arg Thr 770 775 780 Ala Ala Asp Ile Leu Leu Arg Leu Cys Val LeuTrp Asp Asp Leu Gln 785 790 795 800 Ser Ser Val Ile Pro Glu Val Asp ArgGlu Ala Met Leu Lys Pro Asn 805 810 815 Ile Ile Phe Gln Gln Leu Leu PheTrp Thr Asp Pro Arg Asn Leu Val 820 825 830 Asn Gln Thr Gly Ser Thr LysLys Asp Thr Val Gln Leu Thr Phe Leu 835 840 845 Ile Asp Val Leu Lys IleTyr Ala Gln Ile Glu Lys Lys Glu Ile Lys 850 855 860 Lys Met Ile Ile ThrAsn Ile Asn Ala Ile Phe Leu Ser Ser Glu Gln 865 870 875 880 Asp Tyr SerThr Leu Lys Glu Leu Leu Glu Tyr Ser Asp Asp Ile Ala 885 890 895 Glu AsnAsp Asn Leu Asp Asn Val Ser Lys Asn Ala Leu Asp Lys Leu 900 905 910 ArgAsn Asn Leu Asn Ser Leu Ile Glu Glu Ile Asn Glu Arg Ser Glu 915 920 925Thr Gln Thr Lys Asp Glu Asn Asn Thr Ala Asn Asp Gln Tyr Ser Ser 930 935940 Ile Leu Gly Asn Ser Phe Asn Lys Ser Ser Asn Asp Thr Ile Glu His 945950 955 960 Ala Ala Asp Ile Thr Asp Gly Asn Asn Thr Glu Leu Thr Lys ThrThr 965 970 975 Val Asn Ile Ser Ala Val Asp Asn Thr Thr Glu Gln Ser AsnSer Arg 980 985 990 Lys Arg Thr Arg Ser Glu Ala Glu Gln Ile Asp Thr SerLys Asn Leu 995 1000 1005 Glu Asn Met Ser Ile Gln Asp Thr Ser Thr ValAla Lys Asn Val Ser 1010 1015 1020 Phe Val Leu Pro Asp Glu Lys Ser AspAla Met Ser Ile Asp Glu Glu 1025 1030 1035 1040 Asp Lys Asp Ser Glu SerPhe Ser Glu Val Cys 1045 1050 <210> SEQ ID NO 15 <211> LENGTH: 3800<212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE:15 gaaaattcta ctcgacttta ttttgttctt ttgatcttat ttcttttact ttaattatat 60ataggatatt ttcttaggat aattgtaata aaatgcatta atagttttat atgtccattt 120ctggtgcatt actggacata atttcagttc ctttcaccaa agaatttata gccttggaat 180atttccataa tttgtcccac cgggtaatgt gcgtaatgca ataacatata gcatctatat 240ctggtaagcg tagaagaatt aacaaaccta agaataaaat acaaatatgt ttacaaccaa 300aaaatcaact taaatagatg aagagctcgc atatcaattt taacagacct cgctgaaaga 360ctctgaatcc ttatcttctt catctatgga cattgcatct gatttctcgt caggtaaaac 420aaaacttaca ttttttgcta cagttgacgt gtcttgaata ctcatgtttt ccaggttttt 480ggatgtgtca atttgctccg cttctgatct cgttcttttc cttgagttac tttgctctgt 540tgtattgtca actgccgaaa tattaacagt tgttttagtc aattctgtgt tatttccatc 600agttatatca gcagcgtgtt ctatggtgtc atttgaagat ttattgaatg aattccccaa 660aatagacgag tattggtcat tcgcagtgtt gttctcatct tttgtctgag tttctgacct 720ttcattgatc tcttcaatca gcgaattcaa attattcctt agcttgtcca gagcattttt 780gctaacattg tctaaattat cattttctgc aatatcgtca gaatactcaa gaagttcttt 840caaagtagaa taatcttgtt cagaagaaag aaatatagcg tttatattag tgatgatcat 900cttctttatt tctttcttct caatttgagc gtatattttg agcacatcga tcaagaatgt 960aagctgcact gtatcttttt ttgttgaacc tgtctggtta actaagttac gtggatcagt 1020ccaaaatagc aactgttgaa atattatgtt aggctttagc atagcttcac ggtctacctc 1080aggtattaca gagctctgta aatcgtccca aagaacacat agtcttaaga gtatgtccgc 1140agccgtccta gacatacgtt gttgatgagc aggatgtgaa aaacaataga ctggaatgca 1200gaaggcaaat gcctgtacca gcgcttcgtt tgaggaattt atcggcgaaa aatatgacaa 1260aaccaacgtt tcaaacaaat catcatcagt gaacacgtct gccaaaaata gtttgcataa 1320accctccgct gctatcacct gacattccgg taaaccgtta ttctttaaaa ccttgtaaaa 1380tattttgtgc aacgagattg agtcaacttt gccttctccg tctaccacag tgttcccatg 1440tacggaaaaa atatctacaa tgacttgtaa cgcaatatac tttaatgatg cattaccttt 1500cgaaacgcac atacctaaga tgtacatgtt atcaatagcc aacttcacat ccaagagaca 1560acataaacca aggttcttga caccaagctc cctaatattt ggcgcggtat ttctaaccgc 1620tggtgtgatc aaagtgtcca tcaacgacgc aattaaaatg ttttctgtta acggtgtgtt 1680aactagttct agcatatatg atgaccttgt aagacagaga acaattgttg ctgaggacgc 1740ttccttttcg ggtgggagat tatttattat gtcactctca gatacgttgc cgtttccctg 1800cactaagttt tctacagcag aatggaagga tgaaatattg tcgtcttctt catcctcgtt 1860aacttcgtca ttatgtgtgc cgttttcgtc ctcttcatcg acggaagtct catttctgcg 1920attaatcttc ttgcttttta ttttctcttc ttgttcttgt ttttcaatat cgtcgtctct 1980aatatcatta atgatttcta ttgccattgt tacaaaatct ttttcattta tggacagact 2040tttcattaca cgaataccaa ttttaataag aggttcggag agtgtagtta aggccagcat 2100atttcgtacc actgtaagca tcgatctcct tccaacctca tcgctataat catacctttc 2160ggcggcaatc gatagttgct caataataaa ctctaaagtg ttataatcaa aatgcgactg 2220agagtcatta gaaatgtcgt tgtgatgata tctgagaaga atataatggt ttaaatgctc 2280ggataatttt gaggcttctg gaaagttttc ttccagcatt tctgttatat tattatccaa 2340acagtacaaa taaatagccc gaaagaggaa ggcaatttct acggtaaagt ctttccaaat 2400actttcagga aatttgattt tagataatat atctggcctt gattgaaaaa gtgcttctat 2460agccttaaca cacactgagg atcttgagac atccaatttt tctagtaatt ctatcaaatc 2520gccatccaga gcatttaacc aatcatgagc aatgagtctc ttacacgcat ttctcactga 2580taattcccta tcttctaaac cccactcaat caattgatca aaaatatgcg gctcaatatc 2640atcgaaacac tttcttccca ttgatttcaa aattctcgaa tacacgagcc ttctattaac 2700gatgtttaca tctctagccc tctccaagat atacggtcta gtattattat cattgatcaa 2760attcagcatt gcagccctcc gtacttcagc tgacggatcg ttctggatag aagcaactag 2820agttctcgta gcttcaaaat tttcttcatt atcagaaagc tcagttaaat gttcagtttg 2880ctcttcatcc tgaaatttag ttaaacaaaa cacagcctgt atcctaaccg ttggttctct 2940atcataaatc ctcttattta aagacaatat taataaattg aaaagtgatt catcgatttc 3000ccctatatta tccattataa cggctaataa ctgtaaaact ctaaatctga cgttcttgtc 3060agggctttcc acaccacgca aaacatgtct tatgaactga tcgacgaacc ttgagaatat 3120cccttcttca tcattcgtga gcttatagtt ttgttttttg gccaatatca actccctttc 3180taaagaagct ataaatgcag ctactaactt tactatcctg tctccgataa tctcattctt 3240tttcagagga aggatcttag taactaattt atcgaaccaa aagttaaaag catcttcata 3300gccttgctca acagcctttg actgaatttt cttcaaaact gctatatgct tcctgtgtcc 3360tgcataagaa ccctgtgcct tttgaaatac ttcagcaact gagttaaaga ttttcgtatt 3420aatgtcaata ccatcaggat cttgcatgat gggcgcggct gttaacttct ttgtcttatc 3480caaggctgta ggcattactg aagtgttttt gtttgatctt caaagagcta aaaaggtaaa 3540caaacacgaa ggctctaccc tttcatatgc ggtgcacgga ttttaaaagt cgcaataact 3600caagaatacg atagatgcaa taaatcagag gtaaagtgct ttaggctaat actatgagca 3660attcagtata acctgcatta tctccttgtt ttcctttgta tcccccatga tttttttttc 3720ttgttattcc tcttgatacc atattaggca ggattttttt caaaaaacca aatgaaaaat 3780tttcagactg acaagaaaaa 3800 <210> SEQ ID NO 16 <211> LENGTH: 2156 <212>TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (543)...(1727) <400> SEQUENCE: 16tcttttggtg tcaatggtgt attattccga gttactccag gctaggttca ggagtaccaa 60gaatgtactt tatttattta tacaccggag caagtcatat aattacgcaa acgattcgaa 120attgttaaaa gcaggatcaa cgtatctcat ttctttttga aagacgggta atagaaagtc 180tctgagtcgc accccacatg gatatcgtac tattcgtata tggaatgtaa aatactcgca 240atacgatttt atttagcttc acaatctctc aaacttatcg tcttgatcaa tctttacgtt 300ttaccaaata atcgcctgtt tctggccatt ttttgcttat accatctacc atactcgctg 360tccatatgtg acggtgtcgt ctccaagaaa aataacaatg taaattgacc cagcgtgacg 420acagtagact gtaagttata gtacaatcat actctacctt agtcactgtt cctccactgt 480taagtagaga gagagagaga gtttaaagtg gagaaggcaa gaaaaagtgc acttattacg 540 taatg gat ccc acc aaa gca ccc gat ttt aaa ccg cca cag cca aat 587 Met AspPro Thr Lys Ala Pro Asp Phe Lys Pro Pro Gln Pro Asn 1 5 10 15 gaa gaacta caa cca ccg cca gat cca aca cat acg ata cca aaa tct 635 Glu Glu LeuGln Pro Pro Pro Asp Pro Thr His Thr Ile Pro Lys Ser 20 25 30 gga ccc atagtt cca tat gtt tta gct gat tat aat tct tcg atc gat 683 Gly Pro Ile ValPro Tyr Val Leu Ala Asp Tyr Asn Ser Ser Ile Asp 35 40 45 gct cct ttc aatctc gac att tac aaa acc ctg tcg tca agg aaa aaa 731 Ala Pro Phe Asn LeuAsp Ile Tyr Lys Thr Leu Ser Ser Arg Lys Lys 50 55 60 aac gcc aac tca agcaac cga atg gac cat att cca tta aat act agt 779 Asn Ala Asn Ser Ser AsnArg Met Asp His Ile Pro Leu Asn Thr Ser 65 70 75 gac ttc cag cca cta tctcgg gat gta tca tcg gag gag gaa agt gaa 827 Asp Phe Gln Pro Leu Ser ArgAsp Val Ser Ser Glu Glu Glu Ser Glu 80 85 90 95 ggg caa tcg aat gga attgac gct act cta cag gat gtt acg atg act 875 Gly Gln Ser Asn Gly Ile AspAla Thr Leu Gln Asp Val Thr Met Thr 100 105 110 ggg aat ttg ggg gta ctgaag agc caa att gct gat ttg gaa gaa gtt 923 Gly Asn Leu Gly Val Leu LysSer Gln Ile Ala Asp Leu Glu Glu Val 115 120 125 cct cac aca att gta agacaa gcc aga act att gaa gat tac gaa ttt 971 Pro His Thr Ile Val Arg GlnAla Arg Thr Ile Glu Asp Tyr Glu Phe 130 135 140 cct gta cac aga ttg acgaaa aag tta caa gat cct gaa aaa ctg cct 1019 Pro Val His Arg Leu Thr LysLys Leu Gln Asp Pro Glu Lys Leu Pro 145 150 155 ctg atc atc gtt gct tgtgga tca ttt tct ccc ata aca tac cta cat 1067 Leu Ile Ile Val Ala Cys GlySer Phe Ser Pro Ile Thr Tyr Leu His 160 165 170 175 ttg aga atg ttt gaaatg gct tta gat gat atc aat gag caa acg cgt 1115 Leu Arg Met Phe Glu MetAla Leu Asp Asp Ile Asn Glu Gln Thr Arg 180 185 190 ttt gaa gtg gtt ggtggt tat ttt tct cca gta agt gat aac tat caa 1163 Phe Glu Val Val Gly GlyTyr Phe Ser Pro Val Ser Asp Asn Tyr Gln 195 200 205 aag cga ggg tta gcccca gct tat cat cgt gtc cgc atg tgc gaa tta 1211 Lys Arg Gly Leu Ala ProAla Tyr His Arg Val Arg Met Cys Glu Leu 210 215 220 gca tgc gag cgg acatca tct tgg tta atg gtt gat gcc tgg gaa tct 1259 Ala Cys Glu Arg Thr SerSer Trp Leu Met Val Asp Ala Trp Glu Ser 225 230 235 tta caa tca agt tataca agg aca gca aaa gtc ttg gac cat ttc aat 1307 Leu Gln Ser Ser Tyr ThrArg Thr Ala Lys Val Leu Asp His Phe Asn 240 245 250 255 cat gaa ata aatatc aag aga ggt gga atc atg act gta gat ggt gaa 1355 His Glu Ile Asn IleLys Arg Gly Gly Ile Met Thr Val Asp Gly Glu 260 265 270 aaa atg ggc gtaaaa atc atg tta ttg gca ggc ggt gat ctt atc gaa 1403 Lys Met Gly Val LysIle Met Leu Leu Ala Gly Gly Asp Leu Ile Glu 275 280 285 tcc atg ggc gagcct cat gtg tgg gct gat tca gac ctg cac cat att 1451 Ser Met Gly Glu ProHis Val Trp Ala Asp Ser Asp Leu His His Ile 290 295 300 ttg ggt aat tatgga tgt ttg atc gtg gaa agg act ggt tct gat gtt 1499 Leu Gly Asn Tyr GlyCys Leu Ile Val Glu Arg Thr Gly Ser Asp Val 305 310 315 agg tcc ttc ttgctt tcc cat gat atc atg tat gaa cac aga aga aat 1547 Arg Ser Phe Leu LeuSer His Asp Ile Met Tyr Glu His Arg Arg Asn 320 325 330 335 atc ctt attatc aaa caa ctt att tac aat gat att tcc tct acg aaa 1595 Ile Leu Ile IleLys Gln Leu Ile Tyr Asn Asp Ile Ser Ser Thr Lys 340 345 350 gtg cgg cttttc atc aga cgt gga atg tca gtt caa tat ctt ctt cca 1643 Val Arg Leu PheIle Arg Arg Gly Met Ser Val Gln Tyr Leu Leu Pro 355 360 365 aac tct gtcatc cgt tac atc caa gag tat aat cta tac att aat caa 1691 Asn Ser Val IleArg Tyr Ile Gln Glu Tyr Asn Leu Tyr Ile Asn Gln 370 375 380 agt gaa ccggtc aag cag gtc ttg gat agc aaa gag tgagtttatt 1737 Ser Glu Pro Val LysGln Val Leu Asp Ser Lys Glu 385 390 395 acaactctga tactgcagca gttcaaatttaccactttcc tcttcaaggt gcatagaaaa 1797 aaagttcctg gatgcacgat ttaaaatgtttacagcagag caacaatcat gtgaacaatg 1857 tcaaacattt attttaacac ttaataattataatataacc acaccagcgg taagtttcat 1917 aaggaaaacc tttcagacaa acattccagtgaatcgtata cgtaaatcag caaaattagc 1977 ttataaaata cagaatccga agatacttgatctactcgcg ttactattaa tgcgggtaat 2037 gatctatatt gaattttgca cgtctatagtaacttaaaag tcttgtaata tttgaagtaa 2097 caatgccgta taatactgca taatagccctatcaatcgga atataccaaa acatccttt 2156 <210> SEQ ID NO 17 <211> LENGTH:395 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400>SEQUENCE: 17 Met Asp Pro Thr Lys Ala Pro Asp Phe Lys Pro Pro Gln Pro AsnGlu 1 5 10 15 Glu Leu Gln Pro Pro Pro Asp Pro Thr His Thr Ile Pro LysSer Gly 20 25 30 Pro Ile Val Pro Tyr Val Leu Ala Asp Tyr Asn Ser Ser IleAsp Ala 35 40 45 Pro Phe Asn Leu Asp Ile Tyr Lys Thr Leu Ser Ser Arg LysLys Asn 50 55 60 Ala Asn Ser Ser Asn Arg Met Asp His Ile Pro Leu Asn ThrSer Asp 65 70 75 80 Phe Gln Pro Leu Ser Arg Asp Val Ser Ser Glu Glu GluSer Glu Gly 85 90 95 Gln Ser Asn Gly Ile Asp Ala Thr Leu Gln Asp Val ThrMet Thr Gly 100 105 110 Asn Leu Gly Val Leu Lys Ser Gln Ile Ala Asp LeuGlu Glu Val Pro 115 120 125 His Thr Ile Val Arg Gln Ala Arg Thr Ile GluAsp Tyr Glu Phe Pro 130 135 140 Val His Arg Leu Thr Lys Lys Leu Gln AspPro Glu Lys Leu Pro Leu 145 150 155 160 Ile Ile Val Ala Cys Gly Ser PheSer Pro Ile Thr Tyr Leu His Leu 165 170 175 Arg Met Phe Glu Met Ala LeuAsp Asp Ile Asn Glu Gln Thr Arg Phe 180 185 190 Glu Val Val Gly Gly TyrPhe Ser Pro Val Ser Asp Asn Tyr Gln Lys 195 200 205 Arg Gly Leu Ala ProAla Tyr His Arg Val Arg Met Cys Glu Leu Ala 210 215 220 Cys Glu Arg ThrSer Ser Trp Leu Met Val Asp Ala Trp Glu Ser Leu 225 230 235 240 Gln SerSer Tyr Thr Arg Thr Ala Lys Val Leu Asp His Phe Asn His 245 250 255 GluIle Asn Ile Lys Arg Gly Gly Ile Met Thr Val Asp Gly Glu Lys 260 265 270Met Gly Val Lys Ile Met Leu Leu Ala Gly Gly Asp Leu Ile Glu Ser 275 280285 Met Gly Glu Pro His Val Trp Ala Asp Ser Asp Leu His His Ile Leu 290295 300 Gly Asn Tyr Gly Cys Leu Ile Val Glu Arg Thr Gly Ser Asp Val Arg305 310 315 320 Ser Phe Leu Leu Ser His Asp Ile Met Tyr Glu His Arg ArgAsn Ile 325 330 335 Leu Ile Ile Lys Gln Leu Ile Tyr Asn Asp Ile Ser SerThr Lys Val 340 345 350 Arg Leu Phe Ile Arg Arg Gly Met Ser Val Gln TyrLeu Leu Pro Asn 355 360 365 Ser Val Ile Arg Tyr Ile Gln Glu Tyr Asn LeuTyr Ile Asn Gln Ser 370 375 380 Glu Pro Val Lys Gln Val Leu Asp Ser LysGlu 385 390 395 <210> SEQ ID NO 18 <211> LENGTH: 2156 <212> TYPE: DNA<213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 18 aaaggatgttttggtatatt ccgattgata gggctattat gcagtattat acggcattgt 60 tacttcaaatattacaagac ttttaagtta ctatagacgt gcaaaattca atatagatca 120 ttacccgcattaatagtaac gcgagtagat caagtatctt cggattctgt attttataag 180 ctaattttgctgatttacgt atacgattca ctggaatgtt tgtctgaaag gttttcctta 240 tgaaacttaccgctggtgtg gttatattat aattattaag tgttaaaata aatgtttgac 300 attgttcacatgattgttgc tctgctgtaa acattttaaa tcgtgcatcc aggaactttt 360 tttctatgcaccttgaagag gaaagtggta aatttgaact gctgcagtat cagagttgta 420 ataaactcactctttgctat ccaagacctg cttgaccggt tcactttgat taatgtatag 480 attatactcttggatgtaac ggatgacaga gtttggaaga agatattgaa ctgacattcc 540 acgtctgatgaaaagccgca ctttcgtaga ggaaatatca ttgtaaataa gttgtttgat 600 aataaggatatttcttctgt gttcatacat gatatcatgg gaaagcaaga aggacctaac 660 atcagaaccagtcctttcca cgatcaaaca tccataatta cccaaaatat ggtgcaggtc 720 tgaatcagcccacacatgag gctcgcccat ggattcgata agatcaccgc ctgccaataa 780 catgatttttacgcccattt tttcaccatc tacagtcatg attccacctc tcttgatatt 840 tatttcatgattgaaatggt ccaagacttt tgctgtcctt gtataacttg attgtaaaga 900 ttcccaggcatcaaccatta accaagatga tgtccgctcg catgctaatt cgcacatgcg 960 gacacgatgataagctgggg ctaaccctcg cttttgatag ttatcactta ctggagaaaa 1020 ataaccaccaaccacttcaa aacgcgtttg ctcattgata tcatctaaag ccatttcaaa 1080 cattctcaaatgtaggtatg ttatgggaga aaatgatcca caagcaacga tgatcagagg 1140 cagtttttcaggatcttgta actttttcgt caatctgtgt acaggaaatt cgtaatcttc 1200 aatagttctggcttgtctta caattgtgtg aggaacttct tccaaatcag caatttggct 1260 cttcagtacccccaaattcc cagtcatcgt aacatcctgt agagtagcgt caattccatt 1320 cgattgcccttcactttcct cctccgatga tacatcccga gatagtggct ggaagtcact 1380 agtatttaatggaatatggt ccattcggtt gcttgagttg gcgttttttt tccttgacga 1440 cagggttttgtaaatgtcga gattgaaagg agcatcgatc gaagaattat aatcagctaa 1500 aacatatggaactatgggtc cagattttgg tatcgtatgt gttggatctg gcggtggttg 1560 tagttcttcatttggctgtg gcggtttaaa atcgggtgct ttggtgggat ccattacgta 1620 ataagtgcactttttcttgc cttctccact ttaaactctc tctctctctc tacttaacag 1680 tggaggaacagtgactaagg tagagtatga ttgtactata acttacagtc tactgtcgtc 1740 acgctgggtcaatttacatt gttatttttc ttggagacga caccgtcaca tatggacagc 1800 gagtatggtagatggtataa gcaaaaaatg gccagaaaca ggcgattatt tggtaaaacg 1860 taaagattgatcaagacgat aagtttgaga gattgtgaag ctaaataaaa tcgtattgcg 1920 agtattttacattccatata cgaatagtac gatatccatg tggggtgcga ctcagagact 1980 ttctattacccgtctttcaa aaagaaatga gatacgttga tcctgctttt aacaatttcg 2040 aatcgtttgcgtaattatat gacttgctcc ggtgtataaa taaataaagt acattcttgg 2100 tactcctgaacctagcctgg agtaactcgg aataatacac cattgacacc aaaaga 2156 <210> SEQ ID NO19 <211> LENGTH: 3343 <212> TYPE: DNA <213> ORGANISM: Saccharomycescerevisiae <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1526)...(2728) <400> SEQUENCE: 19 gtttgaattg tgtttgtgtt agaaatttgtgtgctttaat gttatgttat aatgaaatct 60 tattagattt atttaacgtt tttgctgtgcttataataaa cattacataa taaaaggagt 120 agaagaaagt ggtagagagg agtacaaatctacctgccag aactctctcc ttatatatat 180 ttccagtggt gtctggatta cctacctcaagccataccat atccatacca tatccataaa 240 cgcctacaaa atttctaccc caatccagcagcttctatca ctatctcgta taccaccata 300 ggcaccacca ctgtttgtgt aaatttactcctgagggggg ggtggctcaa cacggtgtag 360 gccttcttcc cgcacaatcc gatgaaaccccacaatcgcc tccgtctctt ccactgtgca 420 cggcgctagc tcaacatctt ccccgccacatttactgtgg caaagaaggt gcataatcta 480 aaaaaacata cgtatgagaa tggaaagggcaagataatat cggaccgtag tgagtcactt 540 gcttttggta ttgcaaccaa ctgccgcccctcttcccgct cttgcaccaa aacgctaaat 600 gcccattgtg atggctcatc caccctcacgacgaagtaag acccggggca caagaaaata 660 cgagatcata acagttcgag tccgtttattgtgtgcggtt ttggtacgct ttttcgtgag 720 gtgtactacc attcatgaga gtcgttttaggagctgtcat gaaagatatg tatcttgttg 780 atgaactgta aaaatttgca gaaattgcgctattccgttt atttcattgt cgattcggtg 840 ttaatattag gggtacaaaa tatactagaagttctccctc gaggatatag gaatgcgcaa 900 atgggcattt gatgtgacac aaaatttggacaatataacg attcattttt agatcgttgt 960 tcaaccgtcc cagtggccga gtggttaaggcgatgcctgc tatttcctca gaaaagcaat 1020 taggcattgg gttttacctg cgcaggttcgaatcctgtct gtgacgcttt ttttaatttc 1080 tttactccat gacaaaagcg gataaaaattcccgcattcg gcgtaaaaaa atccggtttt 1140 ttttttagca ctcgctgttt ttgcctctaccgggtgaaaa atgacgatga agacggctgg 1200 aattgcgctg catccgctta cgtaggatagaacacctaca aagatttacg aactttattg 1260 ctcgaagatt cgctatccat atctttttagtttcccccca tttcacaatg ggataccgtt 1320 gttttttctg taggtacgct ttctcatagttaatagagtc agtaattcat ttcatttttt 1380 gcagaaagga atttcttcac ctaatttagaatttcatcaa catttattgt atctgcatgg 1440 tataacaaat tagaaaaatt tggaagggaaaaaaaaactg ttgcgtcaat tacttatacc 1500 agggatagaa aaaaaaaaag gaaac atggat ccc aca aga gct ccg gat ttc 1552 Met Asp Pro Thr Arg Ala Pro Asp Phe1 5 aaa ccg cca tct gca gac gag gaa ttg att cct cca ccc gac ccg gaa 1600Lys Pro Pro Ser Ala Asp Glu Glu Leu Ile Pro Pro Pro Asp Pro Glu 10 15 2025 tct aaa att ccc aaa tct att cca att att cca tac gtc tta gcc gat 1648Ser Lys Ile Pro Lys Ser Ile Pro Ile Ile Pro Tyr Val Leu Ala Asp 30 35 40gcg aat tcc tct ata gat gca cct ttt aat att aag agg aag aaa aag 1696 AlaAsn Ser Ser Ile Asp Ala Pro Phe Asn Ile Lys Arg Lys Lys Lys 45 50 55 catcct aag cat cat cat cac cat cat cac agt cgt aaa gaa ggc aat 1744 His ProLys His His His His His His His Ser Arg Lys Glu Gly Asn 60 65 70 gat aaaaaa cat cag cat att cca ttg aac caa gac gac ttt caa cca 1792 Asp Lys LysHis Gln His Ile Pro Leu Asn Gln Asp Asp Phe Gln Pro 75 80 85 ctt tcc gcagaa gtg tct tcc gaa gat gat gac gcg gat ttt aga tcc 1840 Leu Ser Ala GluVal Ser Ser Glu Asp Asp Asp Ala Asp Phe Arg Ser 90 95 100 105 aag gagaga tac ggt tca gat tca acc aca gaa tca gaa act aga ggt 1888 Lys Glu ArgTyr Gly Ser Asp Ser Thr Thr Glu Ser Glu Thr Arg Gly 110 115 120 gtt cagaaa tat cag att gct gat tta gaa gaa gtt cca cat gga atc 1936 Val Gln LysTyr Gln Ile Ala Asp Leu Glu Glu Val Pro His Gly Ile 125 130 135 gtt cgtcaa gca aga acc ttg gaa gac tac gaa ttc ccc tca cac aga 1984 Val Arg GlnAla Arg Thr Leu Glu Asp Tyr Glu Phe Pro Ser His Arg 140 145 150 tta tcgaaa aaa tta ctg gat cca aat aaa ctg ccg tta gta ata gta 2032 Leu Ser LysLys Leu Leu Asp Pro Asn Lys Leu Pro Leu Val Ile Val 155 160 165 gca tgtggg tct ttt tca cca atc acc tac ttg cat cta aga atg ttt 2080 Ala Cys GlySer Phe Ser Pro Ile Thr Tyr Leu His Leu Arg Met Phe 170 175 180 185 gaaatg gct tta gat gca atc tct gaa caa aca agg ttt gaa gtc ata 2128 Glu MetAla Leu Asp Ala Ile Ser Glu Gln Thr Arg Phe Glu Val Ile 190 195 200 ggtgga tat tac tcc cct gtt agt gat aac tat caa aag caa ggc ttg 2176 Gly GlyTyr Tyr Ser Pro Val Ser Asp Asn Tyr Gln Lys Gln Gly Leu 205 210 215 gcccca tcc tac cat aga gta cgt atg tgt gaa ttg gcc tgc gaa aga 2224 Ala ProSer Tyr His Arg Val Arg Met Cys Glu Leu Ala Cys Glu Arg 220 225 230 acctca tct tgg ttg atg gtg gat gca tgg gag tca ttg caa cct tca 2272 Thr SerSer Trp Leu Met Val Asp Ala Trp Glu Ser Leu Gln Pro Ser 235 240 245 tacaca aga act gcc aag gtc ttg gat cat ttc aat cac gaa atc aat 2320 Tyr ThrArg Thr Ala Lys Val Leu Asp His Phe Asn His Glu Ile Asn 250 255 260 265att aag aga ggt ggt gta gct act gtt act gga gaa aaa att ggt gtg 2368 IleLys Arg Gly Gly Val Ala Thr Val Thr Gly Glu Lys Ile Gly Val 270 275 280aaa ata atg ttg ctg gct ggt ggt gac cta ata gag tca atg ggt gaa 2416 LysIle Met Leu Leu Ala Gly Gly Asp Leu Ile Glu Ser Met Gly Glu 285 290 295cca aac gtt tgg gcg gac gcc gat tta cat cac att ctc ggt aat tac 2464 ProAsn Val Trp Ala Asp Ala Asp Leu His His Ile Leu Gly Asn Tyr 300 305 310ggt tgt ttg att gtc gaa cgt act ggt tct gat gta agg tct ttt ttg 2512 GlyCys Leu Ile Val Glu Arg Thr Gly Ser Asp Val Arg Ser Phe Leu 315 320 325tta tcc cat gat att atg tat gaa cat aga agg aat att ctt atc atc 2560 LeuSer His Asp Ile Met Tyr Glu His Arg Arg Asn Ile Leu Ile Ile 330 335 340345 aag caa ctc atc tat aat gat att tct tcc acg aaa gtt cgt cta ttt 2608Lys Gln Leu Ile Tyr Asn Asp Ile Ser Ser Thr Lys Val Arg Leu Phe 350 355360 atc aga cgc gcc atg tct gta caa tat ttg tta cct aat tcg gtc atc 2656Ile Arg Arg Ala Met Ser Val Gln Tyr Leu Leu Pro Asn Ser Val Ile 365 370375 agg tat atc caa gaa cat aga cta tat gtg gac caa acc gaa cct gtt 2704Arg Tyr Ile Gln Glu His Arg Leu Tyr Val Asp Gln Thr Glu Pro Val 380 385390 aag caa gtt ctt gga aac aaa gaa tgatttgccg tccggaattg cttcgttctt2758 Lys Gln Val Leu Gly Asn Lys Glu 395 400 tctttcatct ttctctttacaatttccaat tttcccctac aggaattaat tggagggtac 2818 aagcgagtag aaatgtgacatatgacttac ctatctgtgt tttagtatag tttttttttc 2878 tgtagtataa ttcacttttacactaatttt ttcgcctttt tctcttaaag agctaatttc 2938 tataaccttc agcggttataccaaatataa aaaatggaag gaaaacaaac agtaagaaat 2998 aagcgcaaca gcacgttagttcaccattgg attccaacat ttcaaaattt aatctaatgg 3058 caagagatat cacatttttgaccgtatttt tagaaagttg tggcgctgta aataatgatg 3118 aggcaggaaa attgttatctgcttggactt caaccgtacg cattgaggga ccggaatcaa 3178 ccgactctaa ttcattatatattccactgc taccacctgg aatgttgaaa gtatgtttct 3238 cctagcaaaa ttaaaacccatccgtgaatg aagcgttact aactataata actggtagct 3298 ttgtcactcg taccaggaaaagtgaagatt aaactgaatt ttaaa 3343 <210> SEQ ID NO 20 <211> LENGTH: 401<212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE:20 Met Asp Pro Thr Arg Ala Pro Asp Phe Lys Pro Pro Ser Ala Asp Glu 1 510 15 Glu Leu Ile Pro Pro Pro Asp Pro Glu Ser Lys Ile Pro Lys Ser Ile 2025 30 Pro Ile Ile Pro Tyr Val Leu Ala Asp Ala Asn Ser Ser Ile Asp Ala 3540 45 Pro Phe Asn Ile Lys Arg Lys Lys Lys His Pro Lys His His His His 5055 60 His His His Ser Arg Lys Glu Gly Asn Asp Lys Lys His Gln His Ile 6570 75 80 Pro Leu Asn Gln Asp Asp Phe Gln Pro Leu Ser Ala Glu Val Ser Ser85 90 95 Glu Asp Asp Asp Ala Asp Phe Arg Ser Lys Glu Arg Tyr Gly Ser Asp100 105 110 Ser Thr Thr Glu Ser Glu Thr Arg Gly Val Gln Lys Tyr Gln IleAla 115 120 125 Asp Leu Glu Glu Val Pro His Gly Ile Val Arg Gln Ala ArgThr Leu 130 135 140 Glu Asp Tyr Glu Phe Pro Ser His Arg Leu Ser Lys LysLeu Leu Asp 145 150 155 160 Pro Asn Lys Leu Pro Leu Val Ile Val Ala CysGly Ser Phe Ser Pro 165 170 175 Ile Thr Tyr Leu His Leu Arg Met Phe GluMet Ala Leu Asp Ala Ile 180 185 190 Ser Glu Gln Thr Arg Phe Glu Val IleGly Gly Tyr Tyr Ser Pro Val 195 200 205 Ser Asp Asn Tyr Gln Lys Gln GlyLeu Ala Pro Ser Tyr His Arg Val 210 215 220 Arg Met Cys Glu Leu Ala CysGlu Arg Thr Ser Ser Trp Leu Met Val 225 230 235 240 Asp Ala Trp Glu SerLeu Gln Pro Ser Tyr Thr Arg Thr Ala Lys Val 245 250 255 Leu Asp His PheAsn His Glu Ile Asn Ile Lys Arg Gly Gly Val Ala 260 265 270 Thr Val ThrGly Glu Lys Ile Gly Val Lys Ile Met Leu Leu Ala Gly 275 280 285 Gly AspLeu Ile Glu Ser Met Gly Glu Pro Asn Val Trp Ala Asp Ala 290 295 300 AspLeu His His Ile Leu Gly Asn Tyr Gly Cys Leu Ile Val Glu Arg 305 310 315320 Thr Gly Ser Asp Val Arg Ser Phe Leu Leu Ser His Asp Ile Met Tyr 325330 335 Glu His Arg Arg Asn Ile Leu Ile Ile Lys Gln Leu Ile Tyr Asn Asp340 345 350 Ile Ser Ser Thr Lys Val Arg Leu Phe Ile Arg Arg Ala Met SerVal 355 360 365 Gln Tyr Leu Leu Pro Asn Ser Val Ile Arg Tyr Ile Gln GluHis Arg 370 375 380 Leu Tyr Val Asp Gln Thr Glu Pro Val Lys Gln Val LeuGly Asn Lys 385 390 395 400 Glu <210> SEQ ID NO 21 <211> LENGTH: 3343<212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE:21 tttaaaattc agtttaatct tcacttttcc tggtacgagt gacaaagcta ccagttatta 60tagttagtaa cgcttcattc acggatgggt tttaattttg ctaggagaaa catactttca 120acattccagg tggtagcagt ggaatatata atgaattaga gtcggttgat tccggtccct 180caatgcgtac ggttgaagtc caagcagata acaattttcc tgcctcatca ttatttacag 240cgccacaact ttctaaaaat acggtcaaaa atgtgatatc tcttgccatt agattaaatt 300ttgaaatgtt ggaatccaat ggtgaactaa cgtgctgttg cgcttatttc ttactgtttg 360ttttccttcc attttttata tttggtataa ccgctgaagg ttatagaaat tagctcttta 420agagaaaaag gcgaaaaaat tagtgtaaaa gtgaattata ctacagaaaa aaaaactata 480ctaaaacaca gataggtaag tcatatgtca catttctact cgcttgtacc ctccaattaa 540ttcctgtagg ggaaaattgg aaattgtaaa gagaaagatg aaagaaagaa cgaagcaatt 600ccggacggca aatcattctt tgtttccaag aacttgctta acaggttcgg tttggtccac 660atatagtcta tgttcttgga tatacctgat gaccgaatta ggtaacaaat attgtacaga 720catggcgcgt ctgataaata gacgaacttt cgtggaagaa atatcattat agatgagttg 780cttgatgata agaatattcc ttctatgttc atacataata tcatgggata acaaaaaaga 840ccttacatca gaaccagtac gttcgacaat caaacaaccg taattaccga gaatgtgatg 900taaatcggcg tccgcccaaa cgtttggttc acccattgac tctattaggt caccaccagc 960cagcaacatt attttcacac caattttttc tccagtaaca gtagctacac cacctctctt 1020aatattgatt tcgtgattga aatgatccaa gaccttggca gttcttgtgt atgaaggttg 1080caatgactcc catgcatcca ccatcaacca agatgaggtt ctttcgcagg ccaattcaca 1140catacgtact ctatggtagg atggggccaa gccttgcttt tgatagttat cactaacagg 1200ggagtaatat ccacctatga cttcaaacct tgtttgttca gagattgcat ctaaagccat 1260ttcaaacatt cttagatgca agtaggtgat tggtgaaaaa gacccacatg ctactattac 1320taacggcagt ttatttggat ccagtaattt tttcgataat ctgtgtgagg ggaattcgta 1380gtcttccaag gttcttgctt gacgaacgat tccatgtgga acttcttcta aatcagcaat 1440ctgatatttc tgaacacctc tagtttctga ttctgtggtt gaatctgaac cgtatctctc 1500cttggatcta aaatccgcgt catcatcttc ggaagacact tctgcggaaa gtggttgaaa 1560gtcgtcttgg ttcaatggaa tatgctgatg ttttttatca ttgccttctt tacgactgtg 1620atgatggtga tgatgatgct taggatgctt tttcttcctc ttaatattaa aaggtgcatc 1680tatagaggaa ttcgcatcgg ctaagacgta tggaataatt ggaatagatt tgggaatttt 1740agattccggg tcgggtggag gaatcaattc ctcgtctgca gatggcggtt tgaaatccgg 1800agctcttgtg ggatccatgt ttcctttttt tttttctatc cctggtataa gtaattgacg 1860caacagtttt tttttccctt ccaaattttt ctaatttgtt ataccatgca gatacaataa 1920atgttgatga aattctaaat taggtgaaga aattcctttc tgcaaaaaat gaaatgaatt 1980actgactcta ttaactatga gaaagcgtac ctacagaaaa aacaacggta tcccattgtg 2040aaatgggggg aaactaaaaa gatatggata gcgaatcttc gagcaataaa gttcgtaaat 2100ctttgtaggt gttctatcct acgtaagcgg atgcagcgca attccagccg tcttcatcgt 2160catttttcac ccggtagagg caaaaacagc gagtgctaaa aaaaaaaccg gattttttta 2220cgccgaatgc gggaattttt atccgctttt gtcatggagt aaagaaatta aaaaaagcgt 2280cacagacagg attcgaacct gcgcaggtaa aacccaatgc ctaattgctt ttctgaggaa 2340atagcaggca tcgccttaac cactcggcca ctgggacggt tgaacaacga tctaaaaatg 2400aatcgttata ttgtccaaat tttgtgtcac atcaaatgcc catttgcgca ttcctatatc 2460ctcgagggag aacttctagt atattttgta cccctaatat taacaccgaa tcgacaatga 2520aataaacgga atagcgcaat ttctgcaaat ttttacagtt catcaacaag atacatatct 2580ttcatgacag ctcctaaaac gactctcatg aatggtagta cacctcacga aaaagcgtac 2640caaaaccgca cacaataaac ggactcgaac tgttatgatc tcgtattttc ttgtgccccg 2700ggtcttactt cgtcgtgagg gtggatgagc catcacaatg ggcatttagc gttttggtgc 2760aagagcggga agaggggcgg cagttggttg caataccaaa agcaagtgac tcactacggt 2820ccgatattat cttgcccttt ccattctcat acgtatgttt ttttagatta tgcaccttct 2880ttgccacagt aaatgtggcg gggaagatgt tgagctagcg ccgtgcacag tggaagagac 2940ggaggcgatt gtggggtttc atcggattgt gcgggaagaa ggcctacacc gtgttgagcc 3000acccccccct caggagtaaa tttacacaaa cagtggtggt gcctatggtg gtatacgaga 3060tagtgataga agctgctgga ttggggtaga aattttgtag gcgtttatgg atatggtatg 3120gatatggtat ggcttgaggt aggtaatcca gacaccactg gaaatatata taaggagaga 3180gttctggcag gtagatttgt actcctctct accactttct tctactcctt ttattatgta 3240atgtttatta taagcacagc aaaaacgtta aataaatcta ataagatttc attataacat 3300aacattaaag cacacaaatt tctaacacaa acacaattca aac 3343 <210> SEQ ID NO 22<211> LENGTH: 1900 <212> TYPE: DNA <213> ORGANISM: Saccharomycescerevisiae <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(813)...(1853) <400> SEQUENCE: 22 ttctactact ccacgtacaa aaaagagcacgctgctttat ttatactttt gtgccacaag 60 aatgatcaac atcaacataa atatcaactagtatctgcaa cacatctgct ccacggaact 120 aaacccgttg agcagtgccc cgtggaaacgtaaactatcg caaattggga ttaacaagcc 180 aaaaacagcc aagcaagatt cacgaaaccgcgcctcgttt ggaccccgaa ggcccattta 240 acggccggcc gttacaagca agatcggcagagcaaaccac tccccagcac cacagcacat 300 cactgcacga gcaacaataa ctagaacatggcagatagcg aggatacctc tgtgatcctg 360 cagggcatcg acacaatcaa cagcgtggagggcctggaag aagatggtta cctcagcgac 420 gaggacacgt cactcagcaa cgagctcgcagatgcacagc gtcaatggga agagtcgctg 480 caacagttga acaagctgct caactgggtcctgctgcccc tgctgggcaa gtatataggt 540 aggagaatgg ccaagactct atggagtaggttcattgaac actttgtata agtgtttgtt 600 gtttatgtat ccgcatatag cagttataacagataaatgg cacttttcgc acacccgttg 660 ttttatctcc gatagtacgt gggcctttatttatggtcgt ttaacgaaag aacggcatct 720 tgaattgagc aggtatttaa aagataggacgagaaacaag cacatgatct gtgtcgaaaa 780 aaagtagcaa agagaaaaag taggaggata ggatg aac agg aaa gta gct atc 833 Met Asn Arg Lys Val Ala Ile 1 5 gta acgggt act aat agt aat ctt ggt ctg aac att gtg ttc cgt ctg 881 Val Thr GlyThr Asn Ser Asn Leu Gly Leu Asn Ile Val Phe Arg Leu 10 15 20 att gaa actgag gac acc aat gtc aga ttg acc att gtg gtg act tct 929 Ile Glu Thr GluAsp Thr Asn Val Arg Leu Thr Ile Val Val Thr Ser 25 30 35 aga acg ctt cctcga gtg cag gag gtg att aac cag att aaa gat ttt 977 Arg Thr Leu Pro ArgVal Gln Glu Val Ile Asn Gln Ile Lys Asp Phe 40 45 50 55 tac aac aaa tcaggc cgt gta gag gat ttg gaa ata gac ttt gat tat 1025 Tyr Asn Lys Ser GlyArg Val Glu Asp Leu Glu Ile Asp Phe Asp Tyr 60 65 70 ctg ttg gtg gac ttcacc aac atg gtg agt gtc ttg aac gca tat tac 1073 Leu Leu Val Asp Phe ThrAsn Met Val Ser Val Leu Asn Ala Tyr Tyr 75 80 85 gac atc aac aaa aag tacagg gcg ata aac tac ctt ttc gtg aat gct 1121 Asp Ile Asn Lys Lys Tyr ArgAla Ile Asn Tyr Leu Phe Val Asn Ala 90 95 100 gcg caa ggt atc ttt gacggt ata gat tgg atc gga gcg gtc aag gag 1169 Ala Gln Gly Ile Phe Asp GlyIle Asp Trp Ile Gly Ala Val Lys Glu 105 110 115 gtt ttc acc aat cca ttggag gca gtg aca aat ccg aca tac aag ata 1217 Val Phe Thr Asn Pro Leu GluAla Val Thr Asn Pro Thr Tyr Lys Ile 120 125 130 135 caa ctg gtg ggc gtcaag tct aaa gat gac atg ggg ctt att ttc cag 1265 Gln Leu Val Gly Val LysSer Lys Asp Asp Met Gly Leu Ile Phe Gln 140 145 150 gcc aat gtg ttt ggtccg tac tac ttt atc agt aaa att ctg cct caa 1313 Ala Asn Val Phe Gly ProTyr Tyr Phe Ile Ser Lys Ile Leu Pro Gln 155 160 165 ttg acc agg gga aaggct tat att gtt tgg att tcg agt att atg tcc 1361 Leu Thr Arg Gly Lys AlaTyr Ile Val Trp Ile Ser Ser Ile Met Ser 170 175 180 gat cct aag tat ctttcg ttg aac gat att gaa cta cta aag aca aat 1409 Asp Pro Lys Tyr Leu SerLeu Asn Asp Ile Glu Leu Leu Lys Thr Asn 185 190 195 gcc tct tat gag ggctcc aag cgt tta gtt gat tta ctg cat ttg gcc 1457 Ala Ser Tyr Glu Gly SerLys Arg Leu Val Asp Leu Leu His Leu Ala 200 205 210 215 acc tac aaa gacttg aaa aag ctg ggc ata aat cag tat gta gtt caa 1505 Thr Tyr Lys Asp LeuLys Lys Leu Gly Ile Asn Gln Tyr Val Val Gln 220 225 230 ccg ggc ata tttaca agc cat tcc ttc tcc gaa tat ttg aat ttt ttc 1553 Pro Gly Ile Phe ThrSer His Ser Phe Ser Glu Tyr Leu Asn Phe Phe 235 240 245 acc tat ttc ggcatg cta tgc ttg ttc tat ttg gcc agg ctg ttg ggg 1601 Thr Tyr Phe Gly MetLeu Cys Leu Phe Tyr Leu Ala Arg Leu Leu Gly 250 255 260 tct cca tgg cacaat att gat ggt tat aaa gct gcc aat gcc cca gta 1649 Ser Pro Trp His AsnIle Asp Gly Tyr Lys Ala Ala Asn Ala Pro Val 265 270 275 tac gta act agattg gcc aat cca aac ttt gag aaa caa gac gta aaa 1697 Tyr Val Thr Arg LeuAla Asn Pro Asn Phe Glu Lys Gln Asp Val Lys 280 285 290 295 tac ggt tctgct acc tct agg gat ggt atg cca tat atc aag acg cag 1745 Tyr Gly Ser AlaThr Ser Arg Asp Gly Met Pro Tyr Ile Lys Thr Gln 300 305 310 gaa ata gaccct act gga atg tct gat gtc ttc gct tat ata cag aag 1793 Glu Ile Asp ProThr Gly Met Ser Asp Val Phe Ala Tyr Ile Gln Lys 315 320 325 aag aaa ctggaa tgg gac gag aaa ctg aaa gat caa att gtt gaa act 1841 Lys Lys Leu GluTrp Asp Glu Lys Leu Lys Asp Gln Ile Val Glu Thr 330 335 340 aga acc cccatt taatatatct ctgcgtacat atgtatatat atatatgtgt 1893 Arg Thr Pro Ile 345gtatata 1900 <210> SEQ ID NO 23 <211> LENGTH: 347 <212> TYPE: PRT <213>ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 23 Met Asn Arg LysVal Ala Ile Val Thr Gly Thr Asn Ser Asn Leu Gly 1 5 10 15 Leu Asn IleVal Phe Arg Leu Ile Glu Thr Glu Asp Thr Asn Val Arg 20 25 30 Leu Thr IleVal Val Thr Ser Arg Thr Leu Pro Arg Val Gln Glu Val 35 40 45 Ile Asn GlnIle Lys Asp Phe Tyr Asn Lys Ser Gly Arg Val Glu Asp 50 55 60 Leu Glu IleAsp Phe Asp Tyr Leu Leu Val Asp Phe Thr Asn Met Val 65 70 75 80 Ser ValLeu Asn Ala Tyr Tyr Asp Ile Asn Lys Lys Tyr Arg Ala Ile 85 90 95 Asn TyrLeu Phe Val Asn Ala Ala Gln Gly Ile Phe Asp Gly Ile Asp 100 105 110 TrpIle Gly Ala Val Lys Glu Val Phe Thr Asn Pro Leu Glu Ala Val 115 120 125Thr Asn Pro Thr Tyr Lys Ile Gln Leu Val Gly Val Lys Ser Lys Asp 130 135140 Asp Met Gly Leu Ile Phe Gln Ala Asn Val Phe Gly Pro Tyr Tyr Phe 145150 155 160 Ile Ser Lys Ile Leu Pro Gln Leu Thr Arg Gly Lys Ala Tyr IleVal 165 170 175 Trp Ile Ser Ser Ile Met Ser Asp Pro Lys Tyr Leu Ser LeuAsn Asp 180 185 190 Ile Glu Leu Leu Lys Thr Asn Ala Ser Tyr Glu Gly SerLys Arg Leu 195 200 205 Val Asp Leu Leu His Leu Ala Thr Tyr Lys Asp LeuLys Lys Leu Gly 210 215 220 Ile Asn Gln Tyr Val Val Gln Pro Gly Ile PheThr Ser His Ser Phe 225 230 235 240 Ser Glu Tyr Leu Asn Phe Phe Thr TyrPhe Gly Met Leu Cys Leu Phe 245 250 255 Tyr Leu Ala Arg Leu Leu Gly SerPro Trp His Asn Ile Asp Gly Tyr 260 265 270 Lys Ala Ala Asn Ala Pro ValTyr Val Thr Arg Leu Ala Asn Pro Asn 275 280 285 Phe Glu Lys Gln Asp ValLys Tyr Gly Ser Ala Thr Ser Arg Asp Gly 290 295 300 Met Pro Tyr Ile LysThr Gln Glu Ile Asp Pro Thr Gly Met Ser Asp 305 310 315 320 Val Phe AlaTyr Ile Gln Lys Lys Lys Leu Glu Trp Asp Glu Lys Leu 325 330 335 Lys AspGln Ile Val Glu Thr Arg Thr Pro Ile 340 345 <210> SEQ ID NO 24 <211>LENGTH: 1900 <212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae<400> SEQUENCE: 24 tatatacaca catatatata tatacatatg tacgcagagatatattaaat gggggttcta 60 gtttcaacaa tttgatcttt cagtttctcg tcccattccagtttcttctt ctgtatataa 120 gcgaagacat cagacattcc agtagggtct atttcctgcgtcttgatata tggcatacca 180 tccctagagg tagcagaacc gtattttacg tcttgtttctcaaagtttgg attggccaat 240 ctagttacgt atactggggc attggcagct ttataaccatcaatattgtg ccatggagac 300 cccaacagcc tggccaaata gaacaagcat agcatgccgaaataggtgaa aaaattcaaa 360 tattcggaga aggaatggct tgtaaatatg cccggttgaactacatactg atttatgccc 420 agctttttca agtctttgta ggtggccaaa tgcagtaaatcaactaaacg cttggagccc 480 tcataagagg catttgtctt tagtagttca atatcgttcaacgaaagata cttaggatcg 540 gacataatac tcgaaatcca aacaatataa gcctttcccctggtcaattg aggcagaatt 600 ttactgataa agtagtacgg accaaacaca ttggcctggaaaataagccc catgtcatct 660 ttagacttga cgcccaccag ttgtatcttg tatgtcggatttgtcactgc ctccaatgga 720 ttggtgaaaa cctccttgac cgctccgatc caatctataccgtcaaagat accttgcgca 780 gcattcacga aaaggtagtt tatcgccctg tactttttgttgatgtcgta atatgcgttc 840 aagacactca ccatgttggt gaagtccacc aacagataatcaaagtctat ttccaaatcc 900 tctacacggc ctgatttgtt gtaaaaatct ttaatctggttaatcacctc ctgcactcga 960 ggaagcgttc tagaagtcac cacaatggtc aatctgacattggtgtcctc agtttcaatc 1020 agacggaaca caatgttcag accaagatta ctattagtacccgttacgat agctactttc 1080 ctgttcatcc tatcctccta ctttttctct ttgctactttttttcgacac agatcatgtg 1140 cttgtttctc gtcctatctt ttaaatacct gctcaattcaagatgccgtt ctttcgttaa 1200 acgaccataa ataaaggccc acgtactatc ggagataaaacaacgggtgt gcgaaaagtg 1260 ccatttatct gttataactg ctatatgcgg atacataaacaacaaacact tatacaaagt 1320 gttcaatgaa cctactccat agagtcttgg ccattctcctacctatatac ttgcccagca 1380 ggggcagcag gacccagttg agcagcttgt tcaactgttgcagcgactct tcccattgac 1440 gctgtgcatc tgcgagctcg ttgctgagtg acgtgtcctcgtcgctgagg taaccatctt 1500 cttccaggcc ctccacgctg ttgattgtgt cgatgccctgcaggatcaca gaggtatcct 1560 cgctatctgc catgttctag ttattgttgc tcgtgcagtgatgtgctgtg gtgctgggga 1620 gtggtttgct ctgccgatct tgcttgtaac ggccggccgttaaatgggcc ttcggggtcc 1680 aaacgaggcg cggtttcgtg aatcttgctt ggctgtttttggcttgttaa tcccaatttg 1740 cgatagttta cgtttccacg gggcactgct caacgggtttagttccgtgg agcagatgtg 1800 ttgcagatac tagttgatat ttatgttgat gttgatcattcttgtggcac aaaagtataa 1860 ataaagcagc gtgctctttt ttgtacgtgg agtagtagaa1900 <210> SEQ ID NO 25 <211> LENGTH: 59 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer forPCR <400> SEQUENCE: 25 aggaaagtag ctatcgtaac gggtactaat agtaatcttggtctcttggc ctcctctag 59 <210> SEQ ID NO 26 <211> LENGTH: 59 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer for PCR <400> SEQUENCE: 26 tacgcagaga tatattaaatgggggttcta gtttcaacaa tttcgttcag aatgacacg 59 <210> SEQ ID NO 27 <211>LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: primer for PCR <400> SEQUENCE: 27ttaacagccg cgcccatcat gcaagatcct gatggtattg acattctctt ggcctcctct 60 ag62 <210> SEQ ID NO 28 <211> LENGTH: 59 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer forPCR <400> SEQUENCE: 28 gcatatcaat tttaacagac ctcgctgaaa gactctgaatcctcgttcag aatgacacg 59 <210> SEQ ID NO 29 <211> LENGTH: 429 <212> TYPE:PRT <213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 29 Thr Leu AlaGlu Glu Asn Met Thr Leu Phe Ile His Cys Tyr Ser Lys 1 5 10 15 Gly HisGlu Asn Leu Gln Val Thr Ala Ile His Ile Leu Cys Asp Met 20 25 30 Leu IleSer His Pro Ser Leu Val Ala Pro Val Thr Gln Ala Asp Lys 35 40 45 Glu ThrVal Ala Pro Pro Ala Phe Gln Lys Pro Leu Leu Lys Val Phe 50 55 60 Ser ArgAla Leu Lys Pro Asn Ser Pro Ala Ser Val Gln Thr Ala Ala 65 70 75 80 AlaThr Ala Leu Ser Lys Leu Leu Leu Thr Gly Val Phe Thr Pro Ser 85 90 95 AlaAla Asn Ile Pro Asp Ala Ile Gln Glu Phe Asn Gln His Ala Ile 100 105 110Glu Thr Leu Leu Gln Ser Leu Val Val Ser Phe Phe His Pro Arg Thr 115 120125 Arg Glu Asn Pro Ala Leu Arg Gln Ala Leu Ala Tyr Phe Phe Pro Val 130135 140 Tyr Cys His Ser Arg Pro Asp Asn Thr Gln His Met Arg Lys Ile Thr145 150 155 160 Val Pro Val Ile Arg Thr Ile Leu Asn Ser Ala Glu Glu TyrTyr Ser 165 170 175 Leu Glu Ala Glu Glu Asp Ser Asp Gly Asp Ile Asp GluSer Val Gly 180 185 190 Glu Lys Glu Leu Lys Ala Leu Met Ser Gly Val LeuGly Met Leu Ala 195 200 205 Glu Trp Thr Asp Glu Arg Arg Val Ile Gly LeuGly Gly Glu Arg Val 210 215 220 Leu Ala Gly Gly Leu Ala Ser Ser Asn ValCys Gly Ile Ile His Leu 225 230 235 240 Gln Leu Ile Lys Asp Ile Leu GluArg Val Leu Gly Ile Ser Glu Gly 245 250 255 Ser Asn Arg Cys Ser Lys GlnGln Arg Lys Leu Leu Phe Ser Leu Met 260 265 270 Ser Lys Leu Tyr Ile AlaPro Pro Thr Ala Leu Ser Arg Ser Ala Ser 275 280 285 Gln Ala Pro Glu AspAsp Ser Phe Arg Ser Ser Val Arg Ser Ser His 290 295 300 Gly Glu Leu AsnPro Glu Asn Leu Ala Leu Ala Gln Glu Val Lys Glu 305 310 315 320 Leu LeuAsp Gln Thr Ile Glu Glu Gly Val Ala Ala Asp Ala Ala Ser 325 330 335 ArgAsn Ala Leu Val Lys Val Lys Asn Val Val Leu Lys Leu Leu Ala 340 345 350Ala Pro Met Arg Pro Ser Ser Ala Arg Gly Arg Glu Ser Ser Val Glu 355 360365 Ser Asp Ile Gly Ser Val Arg Ser Ser Arg Ser Val Arg Pro Ser Val 370375 380 Glu Pro Gly Phe Gly Arg Arg Gly Val Ser Val Glu Pro Ser Ile Met385 390 395 400 Glu Glu Asp Glu Asn Glu Asp Ser Arg Ala Thr Leu Asp SerArg Met 405 410 415 Thr Val Ile Lys Glu Glu Asp Ala Asp Ala Met Glu Glu420 425 <210> SEQ ID NO 30 <211> LENGTH: 147 <212> TYPE: PRT <213>ORGANISM: Aspergillus nidulans <400> SEQUENCE: 30 Leu Leu Ser Pro ProLeu Val Arg Ala Thr Val Ile Phe Pro Ser Ser 1 5 10 15 Ser Ser Cys ArgSer Arg Leu Lys Tyr Ser Val Ser Cys Ser Asp Leu 20 25 30 Gln Leu Leu ArgAla Asp Thr Leu His Ile Ser Ala Ile Met Thr Glu 35 40 45 Ser Thr Gln GluGln Gly Asn Asp Gly Gln Arg Met Pro Pro Ala Pro 50 55 60 Ala Thr Pro ValGlu Asp Tyr Val Phe Pro Glu Tyr Arg Leu Lys Arg 65 70 75 80 Val Met AspAsp Pro Glu Lys Thr Pro Leu Leu Leu Ile Ala Cys Gly 85 90 95 Ser Phe SerPro Ile Thr Phe Leu His Leu Arg Met Phe Glu Met Ala 100 105 110 Ala AspTyr Val Lys Leu Ser Thr Asp Phe Glu Ile Ile Gly Gly Tyr 115 120 125 LeuSer Pro Val Ser Asp Ala Tyr Arg Lys Ala Gly Leu Ala Ser Ala 130 135 140Asn His Arg 145 <210> SEQ ID NO 31 <211> LENGTH: 91 <212> TYPE: PRT<213> ORGANISM: Aspergillus nidulans <400> SEQUENCE: 31 Ile Ala Met CysGln Arg Ala Val Asp Gln Thr Ser Asp Trp Met Met 1 5 10 15 Val Asp ThrTrp Glu Pro Met His Lys Glu Tyr Gln Pro Thr Ala Ile 20 25 30 Val Leu AspHis Phe Asp Tyr Glu Ile Asn Thr Val Arg Lys Gly Ile 35 40 45 Asp Thr GlyLys Gly Thr Arg Lys Arg Val Gln Val Val Leu Leu Ala 50 55 60 Gly Ala AspLeu Val His Thr Met Ser Thr Pro Gly Val Trp Ser Glu 65 70 75 80 Lys AspLeu Asp His Ile Leu Gly Gln Tyr Gly 85 90 <210> SEQ ID NO 32 <211>LENGTH: 92 <212> TYPE: PRT <213> ORGANISM: Aspergillus nidulans <400>SEQUENCE: 32 Thr Phe Ile Val Glu Arg Ser Gly Thr Asp Ile Asp Glu Ala LeuAla 1 5 10 15 Ala Leu Gln Pro Trp Lys Lys Asn Ile His Val Ile Gln GlnLeu Ile 20 25 30 Gln Asn Asp Val Ser Ser Thr Lys Ile Arg Leu Phe Leu ArgArg Asp 35 40 45 Met Ser Val Arg Tyr Leu Ile Pro Asp Pro Val Ile Glu TyrIle Tyr 50 55 60 Glu Asn Asn Leu Tyr Met Asp Asp Gly Thr Thr Gln Pro ThrAla Asp 65 70 75 80 Lys Gly Lys Thr Arg Glu Glu Pro Ala Pro Ser Asn 8590 <210> SEQ ID NO 33 <211> LENGTH: 68 <212> TYPE: PRT <213> ORGANISM:Aspergillus nidulans <400> SEQUENCE: 33 Ala Lys Ala Ala Leu Arg Arg LysLys Val His Glu Lys Asn Leu Glu 1 5 10 15 Gln Thr Gln Ala Gln Ile ValGln Leu Glu Gln Gln Ile Tyr Ser Ile 20 25 30 Glu Ala Ala Asn Ile Asn HisGlu Thr Leu Ala Ala Met Lys Ala Ala 35 40 45 Gly Ala Ala Met Glu Lys IleHis Asn Gly Met Thr Val Glu Gln Val 50 55 60 Asp Glu Thr Met 65 <210>SEQ ID NO 34 <211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM:Aspergillus nidulans <400> SEQUENCE: 34 Asp Lys Leu Arg Glu Gln Gln AlaIle Asn Asp Glu Ile Ala Ile Ala 1 5 10 15 Ile Thr Asn Pro Gly Phe GlyGlu Gln Val Asp Glu Glu Asp Leu Glu 20 25 30 Ala Glu Leu Glu Gly Met GluGln Glu Ala Met Asp Glu Arg Met Leu 35 40 45 His Thr Gly Thr Val Pro ValAla Asp Gln Leu Asn Arg Leu Pro Ala 50 55 60 Pro Ala Asn Ala Glu Pro 6570 <210> SEQ ID NO 35 <211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:Aspergillus nidulans <400> SEQUENCE: 35 Ala Lys Ala Lys Gln Lys Ala GluGlu Glu Asp Glu Glu Ala Glu Leu 1 5 10 15 Glu Lys Leu Arg Ala Glu MetAla Met 20 25

What is claimed is:
 1. An isolated nucleic acid or an allelic variantthereof encoding: an AN97 polypeptide comprising the amino acid sequenceof SEQ ID NO:1, as depicted in FIG. 1; an AN80 polypeptide comprisingthe amino acid sequence of SEQ ID NO:3, as depicted in FIG. 2; an AN85polypeptide comprising the amino acid sequence of SEQ ID NO:5, asdepicted in FIG. 3; or an AN17 polypeptide comprising the amino acidsequence of SEQ ID NO:7, as depicted in FIG.
 4. 2. An isolated nucleicacid comprising a sequence selected from the group consisting of: (a)SEQ ID NO:2, as depicted in FIG. 1, or degenerate variants thereof; (b)SEQ ID NO:2, or degenerate variants thereof, wherein T is replaced by U;(c) nucleic acids complementary to (a) and (b); (d) fragments of (a),(b), and (c) that are at least 15 base pairs in length and whichhybridize under stringent conditions to genomic DNA encoding thepolypeptide of SEQ ID NO:1; (e) SEQ ID NO:4, as depicted in FIG. 2, ordegenerate variants thereof; (f) SEQ ID NO:4, or degenerate variantsthereof, wherein T is replaced by U; (g) nucleic acids complementary to(e) and (f); (h) fragments of (e), (f), and (g) that are at least 15base pairs in length and which hybridize under stringent conditions togenomic DNA encoding the polypeptide of SEQ ID NO:3; (i) SEQ ID NO:6, asdepicted in FIG. 3, or degenerate variants thereof; (j) SEQ ID NO:6, ordegenerate variants thereof, wherein T is replaced by U; (k) nucleicacids complementary to (i) and (j); (l) fragments of (i), (j), and (k)that are at least 15 base pairs in length and which hybridize understringent conditions to genomic DNA encoding the polypeptide of SEQ IDNO:5 (m) SEQ ID NO:8, as depicted in FIG. 4, or degenerate variantsthereof; (n) SEQ ID NO:8, or degenerate variants thereof, wherein T isreplaced by U; (o) nucleic acids complementary to (m) and (n); and (p)fragments of (m), (n), and (o) that are at least 15 base pairs in lengthand which hybridize under stringent conditions to genomic DNA encodingthe polypeptide of SEQ ID NO:7.
 3. An isolated nucleic acid fromAspergillus comprising a nucleotide sequence that is at least 85%identical to a nucleotide sequence selected from the group consisting ofSEQ ID NO:2, and encoding an AN97 polypeptide; SEQ ID NO:4, and encodingan AN80 polypeptide; SEQ ID NO:6, and encoding an AN85 polypeptide; andSEQ ID NO:8, and encoding an AN17 polypeptide.
 4. An isolated nucleicacid that is at least 15 base pairs in length and hybridizes understringent conditions to a nucleotide sequence selected from the groupconsisting of SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; and SEQ ID NO:8. 5.An isolated nucleic acid molecule, said molecule comprising the cDNAsequence contained within an American Type Culture Collection (ATCC)accession number selected from the group consisting of ______, ______,______, and ______.
 6. A vector comprising a nucleic acid of claim
 1. 7.A vector comprising a nucleic acid of claim
 2. 8. An expression vectorcomprising a nucleic acid of claim 1 operably linked to a nucleotidesequence regulatory element that controls expression of said nucleicacid.
 9. An expression vector comprising a nucleic acid of claim 2operably linked to a nucleotide sequence regulatory element thatcontrols expression of said nucleic acid.
 10. A genetically engineeredhost cell comprising a nucleic acid of claim
 1. 11. A geneticallyengineered host cell comprising a nucleic acid of claim
 2. 12. A hostcell of claim 10, wherein the cell is a yeast or bacterium.
 13. A hostcell of claim 11, wherein the cell is a yeast or bacterium.
 14. Agenetically engineered host cell comprising a nucleic acid of claim 1operably linked to a nucleotide sequence regulatory element thatcontrols expression of the nucleic acid in the host cell.
 15. A hostcell of claim 14, wherein the cell is a yeast or bacterium.
 16. Agenetically engineered host cell comprising a nucleic acid of claim 2operably linked to a nucleotide sequence regulatory element thatcontrols expression of the nucleic acid in the host cell.
 17. A hostcell of claim 16, wherein the cell is a yeast or bacterium.
 18. Apolypeptide comprising an amino acid sequence selected from the groupconsisting of: the amino acid sequence of SEQ ID NO:1, as depicted inFIG. 1; the amino acid sequence of SEQ ID NO:3, as depicted in FIG. 2;the amino acid sequence of SEQ ID NO:5, as depicted in FIG. 3; and theamino acid sequence of SEQ ID NO:7, as depicted in FIG.
 4. 19. Apolypeptide encoded by the cDNA sequence of the isolated nucleic acidmolecule of claim
 5. 20. A polypeptide encoded by a nucleic acid ofclaim
 2. 21. A polypeptide encoded by a nucleic acid of claim
 3. 22. Amethod for identifying an antifungal agent, the method comprising: (a)contacting an AN polypeptide with a test compound, wherein the ANpolypeptide is selected from the group consisting of AN97, AN17, AN85,and AN80; (b) detecting binding of the test compound to the ANpolypeptide; and (c) determining whether a test compound that binds to 9an AN polypeptide inhibits growth of fungi, relative to growth of fungicultured in the absence of a test compound that binds to an ANpolypeptide, wherein inhibition of growth is an indication that the testcompound is an antifungal agent.
 23. The method of claim 22, wherein theAN polypeptide is derived from a non-pathogenic Aspergillus strain. 24.The method of claim 22, wherein the AN polypeptide is derived from apathogenic Aspergillus strain.
 25. The method of claim 24, wherein thepathogenic Aspergillus strain is selected from the group consisting ofAspergillus fumigatus, Aspergillus flavus, and Aspergillus niger. 26.The method of claim 22, wherein the test compound is immobilized on asubstrate, and binding of the test compound to the AN polypeptide isdetected as immobilization of the AN polypeptide on the immobilized testcompound.
 27. The method of claim 26, wherein immobilization of the ANpolypeptide on the test compound is detected in an immunoassay with anantibody that specifically binds to the AN polypeptide.
 28. The methodof claim 22, wherein the test compound is selected from the groupconsisting of polypeptides, ribonucleic acids, small molecules, anddeoxyribonucleic acids.
 29. The method of claim 28, wherein: (a) the ANpolypeptide is provided as a fusion protein comprising the ANpolypeptide fused to (i) a transcription activation domain of atranscription factor or (ii) a DNA-binding domain of a transcriptionfactor; and (b) the test compound is a polypeptide that is provided as afusion protein comprising the test polypeptide fused to (i) atranscription activation domain of a transcription factor or (ii) aDNA-binding domain of a transcription factor, to interact with the ANfusion polypeptide; and (c) binding of the test compound to the ANpolypeptide is detected as reconstitution of a transcription factor. 30.A pharmaceutical formulation comprising an antifungal agent identifiedby the method of claim 22, and a pharmaceutically acceptable excipient.31. A method for treating an organism having a fungal infection, themethod comprising administering to the organism a therapeuticallyeffective amount of the pharmaceutical formulation of claim
 30. 32. Apharmaceutical formulation comprising an antifungal agent identified bythe method of claim 24, and a pharmaceutically acceptable excipient. 33.A method for treating an Aspergillus infection in an organism, themethod comprising administering to the organism a therapeuticallyeffective amount of the pharmaceutical formulation of claim
 32. 34. Themethod of claim 33, wherein the organism is a rodent or human.
 35. Themethod of claim 33, wherein the organism is a plant.
 36. An antibodythat specifically binds to an AN polypeptide of claim
 20. 37. Anantibody of claim 36, wherein the antibody is a monoclonal antibody. 38.A pharmaceutical formulation comprising an antifungal agent, wherein theagent is a ribozyme that inhibits the function of AN97, AN17, AN80, orAN85.
 39. A method for treating an organism having a fungal infection,the method comprising administering to the organism a therapeuticallyeffective amount of the pharmaceutical formulation of claim
 38. 40. Apharmaceutical formulation comprising an antifungal agent, wherein theagent is an antisense nucleic acid that inhibits the function of AN97,AN17, AN80, or AN85.
 41. A method for treating an organism having afungal infection, the method comprising administering to the organism atherapeutically effective amount of the pharmaceutical formulation ofclaim
 40. 42. A method for identifying an antifungal agent, the methodcomprising: (a) contacting an AN polypeptide with a test compound,wherein the AN polypeptide is selected from the group consisting ofAN97, AN17, AN85, and AN80; (b) detecting a decrease in function of theAN polypeptide contacted with the test compound; and (c) determiningwhether a test compound that decreases function of a contacted ANpolypeptide inhibits growth of fungi, relative to growth of fungicultured in the absence of a test compound that decreases function of acontacted AN polypeptide, wherein inhibition of growth is an indicationthat the test compound is an antifungal agent.
 43. The method of claim42, wherein the test compound is selected from the group consisting ofpolypeptides, ribonucleic acids, small molecules, and deoxyribonucleicacids.
 44. The method of claim 42, wherein the test compound is anantisense oligonucleotide.
 45. The method of claim 42, wherein the testcompound is a ribozyme.
 46. A method for identifying an antifungalagent, the method comprising: (a) contacting a nucleic acid encoding anAN polypeptide with a test compound, wherein the AN polypeptide isselected from the group consisting of AN97, AN17, AN80, and AN85; (b)detecting binding of the test compound to the nucleic acid; and (c)determining whether a test compound that binds the nucleic acid inhibitsgrowth of fungi, relative to growth of fungi cultured in the absence ofthe test compound that binds the nucleic acid, wherein inhibition ofgrowth is an indication that the test compound is an antifungal agent.47. The method of claim 46, wherein the test compound is selected fromthe group consisting of polypeptides, small molecules, ribonucleicacids, and deoxyribonucleic acids.
 48. The method of claim 46, whereinthe test compound is an antisense oligonucleotide.
 49. The method ofclaim 46, wherein the test compound is a ribozyme.
 50. A method foridentifying an anti-yeast agent, the method comprising: (a) contacting ayeast homolog of an AN polypeptide with a test compound, wherein the ANpolypeptide is selected from the group consisting of AN97, AN17, AN85,and AN80; (b) detecting binding of the test compound to the yeasthomolog; and (c) determining whether a test compound that binds to theyeast homolog inhibits growth of yeast, relative to growth of yeastcultured in the absence of the test compound that binds the yeasthomolog, wherein inhibition of growth is indication that the testcompound is an anti-yeast agent.
 51. The method of claim 50, wherein thehomolog is derived from a non-pathogenic yeast strain.
 52. The method ofclaim 51, wherein the homolog is derived from Saccharomyces cerevisiae.53. The method of claim 51, wherein the homolog is selected from thegroup consisting of D9798.4, L8543.16, YGR010W, and L8004.2.
 54. Themethod of claim 50, wherein the homolog is derived from a pathogenicyeast strain.
 55. The method of claim 50, wherein the test compound isimmobilized on a substrate, and binding of test compound to the homologis detected as immobilization of the homolog on the immobilized testcompound.
 56. The method of claim 55, wherein immobilization of thehomolog on the test compound is detected in an immunoassay with anantibody that specifically binds to the homolog.
 57. The method of claim50, wherein the test compound is selected from the group consisting ofpolypeptides, ribonucleic acids, small molecules, and deoxyribonucleicacids.
 58. The method of claim 57, wherein: (a) the homolog is providedas a fusion protein comprising the homolog fused to (i) a transcriptionactivation domain of a transcription factor or (ii) a DNA-binding domainof a transcription factor; and (b) the test compound is a polypeptidethat is provided as a fusion protein comprising the test polypeptidefused to (i) a transcription activation domain of a transcription factoror (ii) a DNA-binding domain of a transcription factor, to interact withthe homolog; and (c) binding of the test polypeptide to the homolog isdetected as reconstitution of a transcription factor.
 59. A method foridentifying an anti-yeast agent, the method comprising: (a) contacting ahomolog of an AN polypeptide with a test compound, wherein the ANpolypeptide is selected from the group consisting of AN97, AN17, AN85,and AN80; (b) detecting a decrease in function of the AN polypeptidecontacted by the homolog; and (c) determining whether a test compoundthat decreases function of a contacted AN polypeptide inhibits growth ofyeast, relative to growth of yeast cultured in the absence of a testcompound that decreases function of a contacted AN polypeptide, whereininhibition of growth is an indication that the test compound is ananti-yeast agent.
 60. The method of claim 59, wherein the test compoundis selected from the group consisting of polypeptides, ribonucleicacids, small molecules, and deoxyribonucleic acids.
 61. The method ofclaim 59, wherein the test compound is an antisense oligonucleotide. 62.The method of claim 59, wherein the test compound is a ribozyme.
 63. Amethod for identifying an anti-yeast agent, the method comprising: (a)contacting a nucleic acid encoding a homolog of an AN polypeptide with atest compound, wherein the AN polypeptide is selected from the groupconsisting of AN97, AN17, AN80, and AN85; (b) detecting binding of thetest compound to the nucleic acid; and (c) determining whether a testcompound that binds the nucleic acid inhibits growth of yeast, relativeto growth of yeast cultured in the absence of a test compound that bindsthe nucleic acid, wherein inhibition of growth is an indication that thetest compound is an anti-yeast agent.
 64. The method of claim 63,wherein the test compound is selected from the group consisting ofpolypeptides, small molecules, ribonucleic acids, and deoxyribonucleicacids.
 65. The method of claim 63, wherein the test compound is anantisense oligonucleotide.
 66. The method of claim 63, wherein the testcompound is a ribozyme.